Translational dysfunction based therapeutics

ABSTRACT

Provided are methods and compositions for inhibiting eukaryotic translation initiation factor. Such methods and compositions may be used alone or in conjunction with other therapies, such as gene therapies, for inhibiting cell proliferation and/or treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in part of PCT/US06/049450, whichclaims priority to U.S. Provisional Patent Applications 60/754,461 filedDec. 28, 2005, 60/794,048 filed Apr. 22, 2006, 60/848,583 filed Sep. 29,2006 and 60/854,404 filed Oct. 25, 2006. This application is also acontinuation-in part of PCT/US07/021167, which claims priority to U.S.Provisional Patent Applications 60/848,583 filed Sep. 29, 2006 and60/854,404 filed Oct. 25, 2006, and 60/942,884 filed Jun. 8, 2007. Thecontents of each of these applications are hereby incorporated herein byreference in their entireties.

GOVERNMENT SUPPORT

The subject invention was made in part with support from the U.S.Government under Grant Numbers CA 88991, PO1 A144236-01, CA 80728, CA98571, S10 RRO 9145, CA 98571, and 807282241 awarded by the NIH andGrant Number DBI-9724504 awarded by the NSF. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND

The eukaryotic translation initiation factor eIF4E (“4E”) is involved inthe modulation of cellular growth. Moderate overexpression of 4E leadsto dysregulated growth and malignant transformation. Both the nuclearand cytoplasmic function of 4E contribute to its ability to transformcells. Overexpression of 4E in vivo results in frank tumor formation,and the onset of tumor formation is greatly enhanced when 4Eoverexpression is placed within the context of a myc mouse background,suggesting again that 4E acts in concert with other oncogenes to promoteneoplastic transformation. 4E is believed to represent one of the sevengenes whose expression, when up-regulated in cancers, is predictive ofmetastatic disease. A variety of studies have been done demonstratingthat existence of elevated 4E activity within surgical margins is a poorprognosis factor.

In the cytoplasm, 4E is required for cap-dependent translation, aprocess highly conserved from yeast to humans. 4E is believed to bindthe methyl-7-guanosine cap moiety present on the 5′ end of mRNAs andsubsequently recruits the given mRNA to the ribosome.

In the nucleus, 4E is a critical node in an RNA regulon that impactsnearly every stage of cell cycle progression. Specifically, 4Ecoordinately promotes the mRNA export, and in some cases alsotranslation, of several genes involved in cell cycle progression. Forexample, 4E functions to promote export from the nucleus to thecytoplasm of at least two mRNAs, cyclin D1 and ornithine decarboxylase(ODC), while having no impact on the nuclear to cytoplasmic transport ofGAPDH or actin mRNAs. Moreover, there is evidence that the mRNA exportfunction of 4E is linked to its oncogenic transformation activity.

Dysregulated expression of tumor suppressors and oncogenes that maintainand enhance the malignant phenotype have been described. Among thesemolecules are tumor suppressors like p53, Rb, and APC and oncogenes suchas myc, cyclin D1 and 4E. Their interaction constitute a network ofself-reinforcing feedback loops wherein inactivation of principalelements can lead to the reversal and at times even the sustained lossof the neoplastic phenotype.

4E is overexpressed in a wide variety of malignant cell lines andprimary human tumors including tumors of the breast, colon, head andneck, thyroid, lung, non-Hodgkin's lymphoma, prostate, cervix, bladderand chronic and acute myelogenous leukemias. Consistently, even moderateoverexpression of 4E in rodent cells leads to deregulated proliferationand malignant transformation.

Despite being essential for growth and survival of eukaryotes by actingat a critical step of cap-dependent translation and recruitingtranscripts to the ribosome as a result of its specific interaction withthe 5′ 7-methylguanosine mRNA cap structure, up-regulation of 4E doesnot increase translation of all cap-dependent transcripts, but only of aspecific subset of 4E-sensitive transcripts.

As much as 70% of 4E is present in the nuclei of mammalian cells, whereit associates with nuclear bodies in a wide variety of organism,including yeast, Xenopus and humans. Here, 4E promotes transport ofmRNAs of a specific subset of transcripts such as cyclin D1, but not ofhousekeeping genes such as B-actin and GAPDH. Post-transcriptionalregulation of gene expression at the level of 4E mediated mRNA transportand translation exhibits different gene specificities, with some genesbeing regulated at the level of transport (e.g. cyclin D1) and some atthe level of translation (VEGF), others at both levels (ODC), and stillyet others at neither level (GAPDH). Binding to the m7G cap is requiredboth for mRNA transport and translation by 4E, both of which contributeto this ability to transform cells.

Past observation indicates that 4E's capacity to discriminate betweencyclin D1 and GAPDH is surprising seeing that the traditional view isthat 4E binds the m7G cap found on all mRNAs regardless of othersequence specific features. Thus, this functional discriminationpresents a conundrum in terms of our understanding of 4E mRNArecognition in the nucleus.

Elevated 4E activity has been observed to mediate selectively thetranslation (but not transcription) of a subset of the total collectionof mRNAs expressed within cells, tissues, organs. Specifically, withincells, tumors and/or cancers where 4E activity is present at elevatedlevels, the translation of mRNA transcripts possessing complex 5′UTRregions is selectively upregulated. The repertoire of genes whosetranslation is thereby upregulated in circumstances where elevated 4Eactivity exists is a who's who of genes known to be involved in theregulation of the cell cycle, angiogenesis, proliferation and the like.

Existing cancer therapies are not effectively targeted/selective,thereby forcing patients to experience significant toxicity and sideeffects and/or they are not capable of addressing a wide range ofcancers; neither are they capable of transforming cancer from a terminaldisease process to one that can be managed long-term as are many othersdiseases (cardiovascular, diabetes to name a few). Existing genetherapeutics are conditional replicating lytic viruses, vectors/virusescontaining RNAs encoding prodrug (aka suicide genes), anti-angiogenicagents, immune regulatory cytokines, tumor suppressors toxins and lyticpeptides. Current gene therapeutic vectors/viruses are dependent uponthe presence of elevated levels of 4E protein for the delivery ofsuicide genes, toxins, lytic peptides and/or proteins and/or processes.

SUMMARY

Current therapeutic methods and clinical treatment paradigms do notprovide for enhanced control for viral oncolysis, enhanced control forvirus or vector replication, or enhanced control for gene therapeuticexpression. Also, there are no methods for providing enhanced efficacyand/or safety of gene therapeutic activities. Alternatives orsupplements to gene therapy, such as small molecule inhibitors of 4Eactivity, do not exist. Furthermore, current diagnostic, segmentationand stratification methodologies do not provide for the enhanceddetection, analysis and therapeutic monitoring of 4E regulon activity.Neither do current methods provide for the identification of therapeuticmethods and clinical treatment paradigms that regulate 4E regulonactivity.

Provided are small molecule inhibitors of mRNA nuclear to cytoplasmictransport and/or protein translational processes. Such inhibitorsselectively target the biological impact of elevated 4E activity, and inparticular, 4E regulon activity, within cells, tissues, tumors and/orcancers.

Further provided herein are compositions comprising gene therapeuticvectors and viruses that, among other things, enhance regulation of mRNAnuclear to cytoplasmic transport and/or mRNA translation. The vectorsand viruses may comprise mRNAs encoding proteins contained within genetherapeutic vector/virus required for vector and/or viral replicationand/or lysis, mRNAs encoding therapeutic proteins required for genetherapeutic activity including but not limited to toxins, lytic peptidesand/or proteins and/or processes and therapeutic proteins including butnot limited to prodrug converting enzymes (aka suicide genes),anti-angiogenic proteins, apoptosis cascade enzymes, tumor suppressors,cytokines and immunologically active proteins, RNAi anti-sense, and thelike.

The small molecule compositions and compositions comprising genetherapeutic vectors and viruses may be used alone or in combination toinhibit elevated 4E activity, in particular, 4E regulon activity, withincells and tissues, particularly cancer and tumor cells and tissues, andin mammals. Further, the small molecule compositions and compositionscomprising gene therapeutic vectors and viruses may be used alone or incombination to inhibit cellular proliferation within cells and tissues,particularly cancer and tumor cells and tissues, and in mammals. Suchcells, tissues and mammals preferably may also possess elevated 4Eactivity and/or elevated 4E regulon activity and/or 4E reguloncomponents.

Such enhanced methods and composition for the treatment of cellproliferative disorders in which there exists elevated 4E activityand/or elevated 4E regulon activity and/or 4E regulon components whereadministration of small molecules and/or gene therapeutic alone or incombination fail to eradicate the cell proliferative disorder or canceror tumor, yet inhibit its continued proliferation and expansion thusproviding either an opportunity for the host immune system to eradicatethe cell proliferative disorder or tumor or cancer; or serving to makethe cell proliferative disorder or tumor or cancer manageable throughthe routine administration of small molecules with or without theperiodic co-administration of additional systemic agents/biologics,and/or the periodic co-administration of any one/more of the genetherapeutics methodologies disclosed herein.

Further provided are enhanced imaging and visualization compositions andmethods for cells, tissues and tumors, for example, those possessingelevated 4E activity. Such enhanced imaging and visualizationcompositions and methods may be used, for example, for the detection ofelevated 4E conditions. Detection of elevated 4E conditions may be usedas method of diagnosing, detecting during surgery, following clinicalcourse of therapeutic efficacy and disease progression/regression.

Regulatory mechanisms described herein coordinately provide for theintegrated regulation of 4E activity within normal and cancerous celltypes. Our work provides insight into these processes and provides forassays and/or screens which can be used to identify second generationtherapeutic regulators of 4E activity that can be used to therapeuticadvantage for the treatment of human cancers. Further, the comparison ofthe relative nuclear/cytoplasmic localization of 4E (absolutedifferences or ratios) and factors revealed herein to provide diagnosticcriteria upon which the use of eIF4E inhibitors (direct/indirect) willbe determined to provide a therapeutic benefit. Moreover,coordinated/multiplexed analysis of human tissue samples/biopsies/tissuearrays directly or post laser capture microscope excision of tumormaterial from slide mounted biopsy materials provide the means ofidentifying human conditions wherein 4E is dysfunctionally regulated viaboth the relative and absolute levels of 4E protein and/or activity aswell as the levels of 4E regulon elements and the absolute levels andphosphorylation status of 4E, 4E-BP1 and the like. Via eitherplasma-based or tissue based approaches, the above procedures areenvisioned to enable both the diagnosis of relevant 4E regulon mediateddisease as well providing a useful mechanism of following thetherapeutic response of individuals to therapies that modulate eIF4E andeIF4E regulon activities.

Accordingly, provided are methods and compositions for theidentification, diagnosis and monitoring of 4E regulon activity and forthe discovery of agents that modulate 4E regulon activity. The methods,compositions and agents may be used alone, in combination with or inconjunction with other therapies for the detection and treatment ofdiseases wherein 4E regulon activity is dysfunctional, includingcellular hypertrophy, cancer, and ischemia reperfusion.

Further provided herein are diagnostic, indication segmentation andstratification, therapeutic and disease monitoring compositions andmethods that provide among other things, for the identification ofconditions and clinical indications in which 4E regulon activity isdysfunctionally regulated. Further methods and compositions fordetection, identification and characterization of agents which modulate4E and 4E regulon activity are provided.

Diagnostic compositions and methods may be used alone or in combinationto identify, detect and monitor 4E regulon activity and their modulationof therapeutic agents. Further, compositions and methods may be usedalone or in composition to identify and characterize compounds andagents that modulate 4E regulon activity including but not restricted tothe modulation of 4E regulon component activities.

Further provided are methods and compositions for the identification,detection and monitoring of 4E regulon activity in conditions where thetherapeutic modulation of 4E activity and 4E regulon activity in afashion or manner that serves to increase 4E and in particular 4Eregulon activity. Such conditions to include but are not limited toischemia reperfusion injury and like conditions.

Further provided are enhanced imaging and visualization compositions andmethods for cells, tissues and tumors, for example, those possessingelevated 4E regulon activity. Such enhanced imaging and visualizationcompositions and methods may be used, for example, for the detection ofelevated 4E conditions. Detection of elevated 4E conditions may be usedas method of diagnosing, monitoring prior, during and afteradministration of small molecule therapeutics alone or in combinationwith additional treatments and/or agents as described herein, monitoringbefore, during and after during surgery, and following clinical courseof therapeutic efficacy and disease progression/regression.

Kits for the practice of the methods are also described herein.

These embodiments of the present invention, other embodiments, and theirfeatures and characteristics will be apparent from the description,drawings, and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Ribavirin, but not Rib4C, binds to the functional m7Gcap-bindingsite of eIF4E with the same affinity as m7G mRNA cap. (a) Normalizedcorrected tryptophan fluorescence intensity quenching and their fits forbinding to Ribavirin to eIF4E wild-type (Filled squares), W73A (Opensquares), W56A (stars), Rib4C to wild-type eIF4E (Filled triangles), RTPto wild-type eIF4E (Filled diamonds), and m7GTP to wild-type eIF4E (Opendiamonds). (b) Apparent dissociation constants in micromolar fornucleoside/nucleotide:eIF4E binding. (c) Western blot of eIF4E remainingbound to m7G-Sepharose upon competition with various concentrations ofm7GTP or RTP. Both m7GTP and RTP lead to 50% reduction of binding at aconcentration of approximately 1 uM. (d) Chemical structures of the ketoforms of m7G, Ribavirin, and Rib4C nucleosides. Note: +, positivecharge; R, ribose.

FIG. 2. Ribavirin specifically disrupts subcellular eIF4E organization.(a) Confocal immuno-fluorescence micrographs of Ribavirin-treated NIH3T3 cells stained with DAPI (chromatin), Nopp140 (Cajal bodies andnucleoli), Sc35 (splicing speckles), and eIF4E (eIF4E nuclear bodies andcytoplasmic eIF4E). (b) Western blots of protein extracts of nuclear (N)and cytoplasmic (C) fractions of Ribavirin-treated NIH 3T3 cells, probedfor nuclear and cytoplasmic eIF4E, and for predominantly nuclear Sc35,and predominantly cytoplasmic β-actin, as controls of fractionation andloading. Ribavirin treatment leads to specific disruption of nucleareIF4E bodies and cytoplasmic retention of eIF4E with an EC₅₀ of 0.1-1μM.

FIG. 3. Ribavirin specifically inhibits eIF4E:mRNA binding, inhibitsnucleocytoplasmic mRNA transport, and depletes levels oftransport-regulated proteins. (a) Northern blots of RNA extracts ofnuclear and cytoplasmic fractions of Ribavirin treated NIH 3T3 cells,which were probed as indicated. U6 small nuclear RNA and tRNALys serveas controls for quality of the fractionation. Ribavirin inhibitsnucleocytoplasmic mRNA transport of cyclin D1, but not B-actin, with anapparent EC50 of ca. 1 uM, as judged from the bar graph quantification(top row). N, nuclear; C, cytoplasmic. This effect was confirmed byusing quantitative real-time PCR (FIG. 4 b). (b) Northern and Westernblots of total extracts of Ribavirin-treated cells, exhibiting depletionof cyclin D1, without affecting transcription, mRNAstability, andprotein synthesis. (c) Western blot of total protein extract ofRib4C-treated cells that were probed for cyclin D1. (a) SemiquantitativeRT-PCR of cyclin D1 mRNA contained in eIF4E purified from the nuclei ofRibavirin-treated cells. Control samples were purified by using IgGantibody (−) instead of antibody specific for eIF4E (+).Semiquantitative PCR of VEGF from cytoplasmic extracts wasimmunopurified as above.

FIG. 4. (a) RNA profiles (A₂₆₀/A₂₈₀) of ribosomal purification fractionsand their cyclin D1, GAPDH, ODC, and VEGF mRNA content as measured usingquantitative RT-PCR and represented using threshold cycle (C_(T)). Errorbars represent ±1σ of three independent experiments. Treatment of cellswith 1 μM Ribavirin has no significant effect on ribosomal loading andinitiation of mRNA translation of cyclin D1 and GAPDH mRNAs. Lower C_(T)values indicate higher mRNA abundance. A C_(T) difference of n isequivalent to n²-fold difference in concentration. Note that whereas thecytoplasmic levels of cyclin D1 mRNA are reduced by Ribavirin treatment,the efficiency of its loading onto polysomes relative to monosomes isunaffected, in contrast to ODC and VEGF mRNAs, whose loading onpolysomes is significantly reduced as compared with monosomal loading.(b) Normalized ratios of nuclear to cytoplasmic (Left) and total (Right)mRNA levels of cyclin D1 (red) and VEGF (green) of NIH 3T3 cells treatedwith various concentrations of Ribavirin, as measured by usingquantitative real-time PCR. Note that 2-fold nuclear retention of cyclinD1 mRNA is evident at 0.1 μM Ribavirin

FIG. 5. (a) Western blot of protein extracts of transfected NIH 3T3cells, probed for eIF4E. (b) Percentage of nonpermeabilized NIH 3T3cells binding annexin V (apoptosis) and those taking up propidium iodide(necrosis), as measured using FACS (left axis), and efficiency oftetrazolium dye reduction (metabolism), as measured using opticaldensity (OD) of formazan (right axis), upon Ribavirin treatment for thesame length of time as foci formation assays (see Examples). Error barsrepresent ±1σ of three independent experiments. Significant effects onviability and metabolism are observed only at Ribavirin concentrationsof 100 μM and greater, consistent with its poisoning of guanosinepathways, such as mRNA misincorporation, only at these high millimolarconcentrations. (c) DNA content histograms as measured using propidiumiodide binding of permeabilized NIH 3T3 cells (left axis) and theircumulative probability distributions (right axis) of untreated cells(blue) and cells treated with 0.1 μM Ribavirin (red). Ribavirintreatment increases the proportion of cells restricted to the G₁ phasefrom 58% to 91%. Oncogenic transformation of NIH 3T3 cells mediated byeIF4E is specifically suppressed by Ribavirin with an apparent EC₅₀ of0.1-1 μM and correlates with G₁ cell-cycle arrest.

FIG. 6. Ribavirin suppresses eIF4E-mediated oncogenic transformation.(a) (Left) Anchorage-dependent foci formation of NIH3T3 cells treatedwith Ribavirin and transfected with empty vector (black dashed line),eIF4EWT (blue line), eIF4E W56A (red line), and cells treated with Rib4Cand transfected with eIF4E WT (black solid line). Error bars represent+/−1 sigma of three independent experiments. Probability of focusformation (Pfocus) is defined as the number of foci formed divided bythe number of cells plated. (Right) Photograph of Giemsa-stained dishesof Ribavirin-treated cells transformed by eIF4E. (b) Colony formation ofprimary humanCD34+ myeloid progenitors isolated from patients with AML(M1, solid circles; M5, solid squares) and normal bone marrow (BM, opensquares), as a function of Ribavirin concentration. Ribavirin reducescolony formation of eIF4E-dependent AML-M5 with an apparent IC50 of ca.1 uM, and with no effect on M1 and normal bone marrow myeloid progenitorcells at this concentration. Note that data are internally normalizedand that absolute colony formation efficiencies of AML myeloidprogenitors are greater than that of BM (data not shown). Error barsrepresent +/−1 sigma of four independent experiments. (c) (Left) Meantumor volume in nude mice engrafted with cells derived from ahypopharyngeal eIF4Edependent tumor, as a function of treatment withdaily 1 uM Ribavirin orally at a dose of 40 ug per kg per day (solidsquares). Error bars represent +/−1 sigma of 10 mice. (Right) Photographof tumors resected after 20 days of treatment.

FIG. 7. Ribavirin and m7G mRNA cap are recognized similarly by eIF4E.(a) 1H, 15N HSQC NMRspectra of eIF4E in the absence (black) and presence(red) of saturating concentrations of m7G nucleoside. Note that of the273 residues of the construct, 207 resonances are observed. (b) 1H, 15NHSQC NMR spectra of eIF4E in the presence of saturating concentrationsof m7G (red) and Ribavirin nucleosides (blue). (c) eIF4E backboneresidues that exhibit (red) and do not exhibit (blue) 1H, 15N HSQC NMRchemical shift perturbation upon binding of Ribavirin and m7G mRNA cap.The difference between conformational rearrangements upon cap binding ofmouse eIF4E observed here and those reported for yeast eIF4E may bebecause of differences between mouse and yeast proteins as well asmicelle binding to yeast eIF4E.

FIG. 8. Ribavirin is a physical mimic of 7-methyl guanosine (m⁷G).Isocontour electrostatic potential molecular surfaces of guanosine, m⁷G,Ribavirin, Rib4C, and tiazofurin bases and their chemical structures,with blue to red color gradient corresponding to gradient of decreasingelectropositive and increasing electronegative potential. Arrowindicates the seventh position in the aromatic ring. R, ribose.

FIG. 9. Apparent binding of Ribavirin to recombinant eIF4E in vitro ismethod and condition dependent. (a) As published previously, 20 uL ofm7GTP-Sepharose (Amersham) was mixed with 1 ug of eIF4E in Buffer B (0.3M NaCl, 0.1 M sodium phosphate at pH 7.5, 10 uM protease free BSA [UBS],0.1% NP-40) with 0.1 mM GTP for 30 min at room temperature. Washed beads(three times with 75 bed volumes) were incubated with 50 uM of compoundsas indicated for 30 min at room temperature. Beads were washed (threetimes with 75 bed volumes) to remove dissociated eIF4E, and eIF4Eremaining bound to beads was resolved using SDS-PAGE, and visualizedusing Western blotting and chemiluminescence. Please note that here thebuffer contained 0.1 mM GTP in order to emphasize the specificity ofRibavirin's competition of m7G:eIF4E binding. Also, here we used afusion of mouse eIF4E with the B1 domain of protein G (G4E), which was akind gift of Gerhard Wagner (Harvard Medical School, Boston, Mass.), asdescribed in Zhou et al. (2001) and Kentsis et al. (2004). (b) Asdescribed by Yan et al. (2005), 1 ug of eIF4E was mixed with 20 uL ofm7GTP-Sepharose (Amersham) in 50 bed volumes of LCB buffer (10 mM HEPESat pH 8.0, 100 mM KCl, 0.2 mM EDTA at pH 8.0), supplemented with 10 uMprotease-free BSA (UBS), 0.1% NP-40, and 0.1 mM GTP, for 20 min atpresumed 4° C., as the experimental temperature was not described (Yanet al. 2005). Washed beads (five times with 50 bed volumes of LCBbuffer) were incubated with 5 bed volumes of 50 uM of compounds asindicated for 20 min at 4° C. Then 20 uL of the supernatant containingdissociated eIF4E was transferred to a new tube, cleared of trace beadscontaining bound eIF4E, and resolved using SDS-PAGE, and visualizedusing Western blotting and chemiluminescence.

FIG. 10. Direct observation of specific binding of Ribavirin to purifiedeIF4E in vitro. Mass spectra were recorded using the AgilentTechnologies 1100 LC/MSD integrated liquid chromatograph singlequadrupole electrospray mass spectrometer (ES-MS) operating in positiveion mode. A solution of 20 uM purified G4E (Zhou et al. 2001; Kentsis etal. 2004) was incubated with a mixture of 80 uM Ribavirin (Calbiochem)and 80 uM GTP (Sigma) in 5% aqueous acetonitrile, 20 mM ammonium acetate(pH 6.5), for 1 min at room temperature. The solution was electrosprayeddirectly at 200 mL/min using nebulizer pressure of 20 psi, dryingnitrogen gas at 200° C. and 10 L/min, and capillary voltage of 4.5 kV.(a) ES-MS spectrum plotting ion abundance in 20 uL of the above mixtureas a function of the mass/charge ratio is shown. An ion of ca. 1740amu/z is labeled, corresponding to a +18 protonation state of apo-G4E(higher peak) and the complex of Ribavirin with G4E (lower peak). (b)Hypermass reconstruction of the spectrum shown above was done accordingto standard methods (De Hoffmann and Stroobant 2001) and contains twospecies of population-weighted mean molecular masses of 31,402 and31,649 Da, corresponding to apo-G4E and G4E bound to Ribavirin (243 Da)with a molecular stoichiometry of 1:1, respectively. Please note thatonly a fraction of total ionized eIF4E appears to be bound to Ribavirinbecause of the differences in ionization efficiencies of the apo- andligand-bound species of eIF4E, wherein ligand binding occurs to thefolded, more native-like, and therefore less ionizable, states (DeHoffmann and Stroobant 2001). Thus, obtaining affinities from massspectrometry data is confounded by these differences in ionization. Forcomparison, we obtained Kd's for eIF4E-Ribavirin of 8.4 uM and foreIF4E-RTP, 0.13 uM, using fluorescence spectroscopy paralleling thosedifferences previously observed for m7-guanosine and m7GTP (Kentsis etal. 2004).

FIG. 11. eIF4E associates with cyclin D1 but not GAPDH mRNA in thenuclear fraction of U2OS or HEK293T cells. (a) U2OS total cell lysateswere immunoprecipitated (IP) with either an eIF4E antibody or mouseimmunoglobulin (IgG) as a control. RNAs were detected by RT-PCR asindicated. Tot represents 0.5% of input RNA. (b) U2OS nuclear lysateswere immunoprecipitated using antibodies to eIF4E (mAb eIF4E), PML (mAbPG-M3), or mouse IgG. RNase A indicates treatment before IP as anegative control. Total and nuclear (nc) represent 5% of RNA input. (c)U2OS nuclear lysates were subjected to SNAAP analysis with eIF4EWT-GSTand mutant (W56A and W73A) fusion proteins. GST only was used as anegative control. Bound RNAs were detected by RT-PCR. Nc represents thepercentage of input as indicated. RT-PCRs for A-C were detected byethidium bromide staining. (d) As a control for cap dependence, theability to compete for eIF4E binding by addition of 50 uM 7GpppG capanalogue or 50 uM GpppG negative control was tested in the nuclearfraction of HEK293T cells. The ability of cyclin D1 and GAPDH mRNA toimmunoprecipitate with eIF4E after treatments as indicated was monitoredby semi-quantitative PCR. (e) Parallel RT-PCR methods to the experimentsin A-C confirm the above results indicating eIF4E selectively bindscyclin D1 in a cap-dependent manner. Relative fold values were ascalculated as described in the Materials and Methods for both cyclin D1and GAPDH mRNAs. (f) K562 nuclear lysates were immunoprecipitated usingantibodies to eIF4E (mAb eIF4E), CBC (pAb CBP80), or mouse IgG; ncrepresent 5% of RNA input. (g) Proteins from immunoprecipitations byeIF4E and CBP antibodies or mouse IgG were analyzed by Western blot(WB). White line indicates that intervening lanes have been spliced out.

FIG. 12. Cyclin D1 but not GAPDH mRNAs colocalize with a subset of eIF4Enuclear bodies. (a) Co-localization of cyclin D1 mRNA with PML and eIF4Eproteins was analyzed in U2OS or NIH3T3 cells. Cyclin D1 mRNA wasdetected using in situ hybridization with a digoxigenin labelednick-translated probe to cyclin D1 (red). Cells were then immunostainedusing an eIF4E mAb conjugated directly to FITC (green) and PML mAb 5E10(blue). (b) The same as in A, except digoxigenin nick-translated probesto GAPDH were used for in situ hybridization. Within these panels,different combinations of overlays of the same micrographs are shown tohighlight the localization of cyclin D1 mRNAs with eIF4E nuclear bodies(see arrows). (c) In situ hybridization for cyclin D1 mRNAs andimmunostaining for eIF4E protein in cyclin D1−/− cells was performed asdescribed above. (d) HEK293T cells contain eIF4E bodies similar in size,number and morphology observed for other cell types. Cells were stainedwith a pAb to eIF4E (Morley and Pain, 1995). Staining with mAb eIF4Egave identical results (not depicted). For all panels, confocalmicrographs represent a single optical section through the plane of thecell.

FIG. 13. eIF4E enhanced nucleocytoplasmic transport of cyclin D1 RNA.(a) Nuclear (n) and cytoplasmic (c) fractions were isolated from NIH3T3cells stably transfected with eIF4E WT, eIF4E mutants (W56A and W73A) orPML and RNAs were detected by Northern blot (NB) as indicated. U6snRNA(nuclear) and tRNALys (cytoplasmic) were used as markers for the qualityof the fractionation. (b) Northern blot analysis of total RNAs isolatedfrom NIH3T3 cells transfected as described in A. Ethidium bromidestained gels in A and B demonstrate the quality of the isolated RNA. (c)eIF4E enhanced mRNA transport leads to up-regulated protein levels ofcorresponding mRNAs. Total cell lysates from NIH3T3 cells transfected asindicated were analyzed for protein content by Western blot (WB). (d)Semi-quantitative PCR indicates that eIF4E overexpression does not altercyclin D1 mRNA stability. Act D indicates hours of actinomycintreatment. Right panel represents decreasing amounts of RNA used inRT-PCR showing that conditions are semi-quantitative. (e) Mutant andwild-type eIF4E proteins still form nuclear bodies. NIH3T3 cellsoverexpressing Xpress-tagged eIF4E wild-type or W73A mutant wereimmunostained with anti-Xpress antibody to detect exogenous eIF4E (red)and or mAb eIF4E directly conjugated to FITC (green) to detect bothendogenous and exogenous protein. The confocal micrograph represents asingle optical section through the cell.

FIG. 14. eIF4E specifically associates with 4E-SE from the 3′UTR ofcyclin D1. (a) Schematic representation of chimeric constructs used inthis study. Full 5′ and 3′UTR and different parts of 3′UTR of humancyclin D1 mRNA were cloned up- or downstream of LacZ, respectively.Numbers represent position of UTR fragments in cyclin D1 mRNA. (b)NIH3T3 cells were transiently transfected with chimeric LacZ constructscontaining UTR-LacZ, LacZ-3′UTR, or LacZ constructs different parts ofcyclin D1 3′UTR. The nuclear fractions of the transfected cells wereimmunoprecipitated with mAb eIF4E or mouse IgG for a control. LacZ andB-actin were detected by semi-quantitative RT-PCR and ethidium bromidestaining (left). Nc indicates the nuclear fraction before IP and is 5%input of nuclear mRNA. For the RT-PCR method (right), relative foldenrichment is shown for the IP eIF4E fraction versus the IP IgG fractionindicating the enrichment of LacZ 3′UTR4 in the JP eIF4E. (c) Sequencealignment of cyclin D14E-SE from ClustalW (Thompson et al., 1994).GenBank/EMBL/DDBJ accession numbers are: human gi: 16950654, mouse gi:6680867 and rat gi: 31377522. GenBank/EMBL/DDBJ accession no. forchicken is from the Ensembl database is gallusGallus|5.14792937-14795000 and gi: U40844.

FIG. 15. 4E-SE is sufficient for eIF4E-mediated mRNA transport. PolyARNA purified from nuclear (n) and cytoplasmic (c) fractions of NIH3T3cells, cotransfected with eIF4E-2Flag and chimeric LacZ constructs (asindicated), were analyzed by (a) semi-quantitative RT-PCR and ethidiumbromide staining (left) or (b) Northern blot (NB). (c and d) Northernblot analysis of polyA RNA purified from nuclear (n) and cytoplasmic (c)fractions of HEK293T cells cotransfected with eIF4E or W56A eIF4E andchimeric LacZ constructs (as indicated). Corresponding aliquots takenbefore polyA RNA purification indicate quality of the fractionations.

FIG. 16. The presence of the 4E-SE correlates with increased LacZprotein levels. (a) Protein levels were analyzed by Western blot (WB) oftotal cell lysates from HEK293T cells transiently cotransfected witheIF4E-2Flag constructs and indicated chimeric LacZ constructs or PML,eIF4E-2Flag, and chimeric LacZ constructs. Note that HEK293T cells haveeIF4E nuclear bodies (FIG. 13 c). (b) Northern blot (NB) analysis oftotal RNA from HEK293T cells cotransfected as indicated. Note that noneof the total RNA levels are altered by any of the transfections. (c)Semi-Q PCR analysis indicates that the presence of the 4E-SE does notdetectably alter LacZ mRNA stability. Act D indicates hours treated withactinomycin D. GAPDH is shown as a loading control.

FIG. 17. The 4E-SE contributes to eIF4E mediated oncogenictransformation. (a) Cyclin D1−/− cells were stably transfected witheIF4E or cotransfected with either the coding region of cyclin D1constructs without the 3′ UTR (cycTrunc), the coding region of cyclin D1with the full-length 3′ UTR (cycFull), and the coding region of cyclinD1 with only the 100 nt 4E-SE (cyc4E-SE), and analyzed foranchorage-dependent foci formation assays. Three independent experimentswere preformed in triplicate and error bars indicate +/−SD. Number offoci are relative to vector control, which was set to 100%. (b) Westernblot analysis (WB) of total cell lysates from cyclin D1−/− cells stablytransfected as indicated, showing increased cyclin D1 protein level incells transfected with constructs containing full-length 3′UTR or 4E-SEcompared with the truncated form lacking the 3′UTR sequence. (c) Resultsof quantitative RT-PCR experiments using endogenous eIF4E in cyclinD1−/− cells. The relative ratios of nuclear (N)/cytoplasmic (C) cyclinD1 mRNA was determined using RT-PCR with the relative standard curvesmethod. Values were normalized to CycFull by setting its ratioarbitrarily to 1. Standard methods were used to propagate SDs from theseexperiments.

FIG. 18. Enhanced mRNA export corresponds to elevated protein levels ofeIF4E sensitive targets. (a) Relative fold difference of mRNAs bound tonuclear eIF4E. mRNAs were immunoprecipitated from untreated nuclearlysates or those treated with m⁷GpppG or GpppG (50 μM). Values representrelative fold±sd (normalized against untreated IP IgG which was set to1). Calculations of fold were carried out using the relative standardcurve method (user bulletin#2 ABI Prism 7700). Relative amounts of thetarget mRNA=10^([C(t)−b]/a) were determined for each PCR reaction.Average values±sd were calculated for each set of triplicates. Averagevalues obtained for the IPs (i.e., average relative amount of IP-edtarget mRNA) were divided by values obtained for 5% nuclear input (i.e.,average relative amount of target mRNA present in the 5% of the amountof nuclear extract used for IP). Obtained values±sd (i.e., avIP/av5%nuclear) were normalized by setting “untreated IgG IP” value to 1. B&C)eIF4E enhanced mRNA transport leads to upregulated protein levels ofcorresponding mRNAs. Total cell lysates from U937 (b) or NIH3T3 (c)cells transfected as indicated were analyzed for protein content bywestern methods. Note that in panel c, where human PML wasoverexpressed, the 5E10 mAb PML antibody only recognizes the human PML,not the endogenous mouse PML.

FIG. 19. A common secondary structure for the 4E-SE that acts as azipcode for eIF4E nuclear bodies. (a) Secondary structure for cyclin D14E-SE (c4E-SE) and Pim-1 4E-SE (p4E-SE) as determined by RNase mappingexperiments. Conserved set of A and U nucleotides (UX₂UX₂A) are yellowhighlighted. Panel (b) shows a sample gel. (c) Mapping of p4E-SE: p4E-SEimmunoprecipitates with eIF4E (upper panel); eIF4E promotes export ofLacZ mRNA that contains minimal p4E-SE (lower panel).Cytoplasmic/nuclear (c/n) values represent relative fold±sd, normalizedto LacZ control, which was set to 1. (d) Co-localization of LacZ-p4E-SE,LacZ-c4E-SE or LacZ transcripts with PML and eIF4E protein was examinedin U2OS cells transfected with LacZ/LacZ-4E-SE. LacZ mRNA was detectedusing in situ hybridization with a biotin labeled nick translated probeto LacZ (red). Cells were then immunostained using an eIF4E mAbconjugated directly to FITC (green) and PML mAb 5E10 (blue).Importantly, LacZ mRNAs containing the 4E-SE from either cyclin D1 orPim-1 co-localize to eIF4E nuclear bodies (see arrows). As we showedpreviously for endogenous cyclin D1 mRNA, there are two populations ofeIF4E nuclear bodies, those that co-localize with LacZ mRNA and thosethat co-localize with PML. Magnification was 100× and 3× (for LacZ andc4ESE) or 4× (p4ESE) digital zoom. Scale bar=10 μM.

FIG. 20. The 4E-SE is required for formation of eIF4E dependentcomplexes. (a) and (b) EMSA analysis indicate that LacZ transcriptswhich contain either the cyclin D1 4E-SE (c4E-SE) or the Pim-14E-SE(p4E-SE) formed high molecular weight complexes in the presence ofnuclear lysates (nc). LacZ transcripts (control) without the 4E-SE didnot form these complexes. Addition of purified murine eIF4E with a 6 kDsolubility tag (m4E) or untagged human eIF4E (h4E) causes partial shiftsrelative to shifts observed with nc lysate. With nuclear lysatesimmunodepleted of eIF4E (dpl nc), gel shifts were not observed. Thesecomplexes could be supershifted by an anti-eIF4E antibody (nc+α4E). (c)Mutation of the Pim-14E-SE (p4E-SE) reduces the efficacy of the gelshift. (a) UV cross-linking studies showed formation of specificcomplexes in the 75-90 kD mass range (indicated by arrows). Thesecomplexes are specifically depleted in the presence of excess m⁷GpppGcap (cap) or if lysates are immunodepleted of eIF4E (dpl nc). *indicates complex that is cap and 4E-SE independent. (e) Addition ofribo-oligonucleotide corresponding to the p4E-SE complexes in thepresence of nuclear lysates indicates that this element can efficientlycompete for complex formation. All transcripts were capped and 3′ endlabeled.

FIG. 21. Export of 4E-SE containing mRNAs is independent of ongoing RNAand protein synthesis, and the pathway is saturated by excess 4E-SE. (a)Quantitative real time PCR analysis of mRNA export of LacZ-c4E-SE andLacZ in eIF4E overexpressing cells is shown. Cytoplasmic/nuclear (c/n)values represent relative fold±sd, normalized to LacZ untreated control,which was set to 1. Treatments: actinomycin D (10 μg/ml) for 1 hour;cycloheximide (100 μg/ml) for 1 hour. (b) and (c) LacZ mRNA export wasmonitored as a function of both time and expression of LacZ transcripts−/+4E-SE, induced with doxicycline. Expression as a function of time isshown. In parallel, the extent of export was monitored as the ratio ofc/n mRNA for each case. Full lines represent trends in cells expressingLacZ-c4E-SE; dotted lines are for cells expressing LacZ-p4E-SE.Endogenous mRNAs from the same samples were also examined. Cyclin D1mRNA export was reduced in cells expressing either LacZ-c4E-SE orLacZ-p4E-SE. Importantly, VEGF, which does not contain a 4E-SE, did nothave its export affected in either case. Clearly, as the amount of 4E-SEcontaining mRNAs increases in the cell (C), the ability to export theseis reduced presumably because the 4E-SE dependent export was saturated(b). c/n values represent relative fold±sd, normalized to LacZ only foreach time point. For total RNAs, values represent relative fold±sd,normalized to the fist time point of induction for each transcript (4h), which was set to 1. Average values of LacZ mRNA obtained for eachtime point were normalized by GAPDH mRNA values obtained for the samesample.

FIG. 22. eIF4E dependent export is NXF1 independent and CRM1 dependent.(a) Comparison of LacZ mRNA in the NXF1 IP fractions. Cells werecotransfected with FlagNXF1/Flagp15 and LacZ or LacZ-c4E-SE.Immunoprecipitations were done with anti-Flag-antibody. LacZ/LacZ-c4E-SEmRNA was monitored by real-time PCR and normalized to IgG controls (asdescribed in FIG. 18 a). (b) NXF1 siRNA treatment (72 h) inhibits exportof LacZ but not LacZ-c4E-SE containing mRNAs. The c/n ratios of LacZ orLacZ-c4E-SE mRNAs in cells overexpressing eIF4E, as a function of siRNAtreatment are shown. C/N values represent relative fold±sd, normalizedto LacZ untreated control, which was set to 1. LacZ mRNAs levels werenormalized to 18S rRNA, whose c/n ratio is unaffected by NXF1 siRNA. (c)Western blot (WB) analysis indicates that NXF1 protein levels arereduced by siRNA treatment but not by scrambled controls (DS(-control)).A WB for eIF4G is shown as a negative control. LacZ protein levelscorrespond to alterations in mRNA export shown in panel b. (d)Dependence of c4E-SE export on leptomycin B (LMB). The c/n ratio ofLacZ-c4E-SE mRNA in eIF4E overexpressing U2OS cells indicated that 4E-SEexport was sensitive to LMB (10 ng/ml for 4 h), while LacZ was notsignificantly so. 18S rRNA export was inhibited by LMB as expected,while β-actin mRNA export was not affected by LMB treatment, asexpected. C/N values represent relative fold±sd, normalized to LacZuntreated control, which was set to 1. All RNAs were normalized to GAPDHmRNA.

FIG. 23. Schematic representation of mechanisms for export of differentclasses of RNA. Overview of characteristic features delineating exportof mRNAs via CRM1 or NXF1/p15 pathways are shown together with featuresof eIF4E mediated export of mRNAs.

FIG. 24. A model of the eIF4E RNA regulon. (a) The nuclear compartmentis shaded gray. In the nucleus both cap binding proteins, eIF4E and thecap binding complex CBC are shown. Bulk mRNA export is depicted by mRNAsbound to the CBC and exiting the nucleus in an NXF1 dependent way. mRNAsare depicted as black lines with black balls denoting the 5′ m⁷G cap.mRNAs with the 4E-SE (in green) can be exported in a CRM1 dependentmanner. Once in the cytoplasm, mRNAs with highly structured 5′ UTRs(shown in red) are preferentially translated in an eIF4E dependentmanner. Coloured boxes correspond to the level of control shown in partB. (b) Schematic representation of the regulon showing that eIF4Eimpacts on mRNA export and translation depending on the presence of thecorrect USER code. Example RNAs for each level of modulation are given.Below, example cellular regulators are given. Finally, compounds thatmodulate the given step in the regulon are shown. Although many RNAsfall into each category and many other regulators and compounds mayexist at each step of control, we have only given examples for the sakeof clarity. Further, the position of regulators and compounds in thisdiagram does not preclude any other unrelated activities these may havein the cell, but simply refers to their currently known role in theeIF4E regulon.

FIG. 25 depicts unsupervised hierarchical clustering analysis of proteinexpression level in breast cancers performed using AQUA scores forestrogen receptor (ER), progesterone receptor (PR), epidermal growthfactor receptor (EGFR), Her2, and eIF4E. For additional details, seeExample 5.

FIG. 26 depicts expression analysis performed from bulk tumor RNAextracted from 141 primary breast cancers and run on Affymetrix U133plus 2.0 arrays (Andrea Richardson Dana-Farber Cancer Institute). Theanalysis was performed using hierarchical clustering function of dChipsoftware. The genes displayed are those that are differentiallyexpressed (at least 1.5 fold with 90% confidence) between tumors withhigh expression (>2 fold above mean) of 4E compared to tumors with lowexpression (>2 fold below mean).

FIG. 27 shows that elevated expression of 4E alters the activity of Akt1and downstream effectors. (A) Western blot analysis of whole-cellextracts from cells over-expressing 4E wt or mutants in MEF Akt1 wt and−/− lines. Proteins detected are as indicated. βactin is shown as aprotein loading control. (B) Western blot analysis of whole-cellextracts from MEF Akt1 wt derived cells treated with the PI3K inhibitor,LY294002 (LY; 50 μM for 1 hr). Proteins detected are as indicated. GAPDHis shown as a protein loading control.

FIG. 28 shows that Akt 1 is required for 4E mediated apoptotic rescue ofserum starved cells. Panels shown are representative fields from TUNELexperiments (blue—DAPI (viable), red—apoptotic; see Materials andMethods) of 4E over-expressing cells derived from MEF Akt1 wt and −/−lines. Scale bar is set at 50 μm. Graphs represent quantitativemeasurements by flow cytometry of apoptosis using Annexin V (Ann.V) andpropidium iodide (PI) staining of indicated cells (see Materials andMethods). Bar color is as follows: Ann.V−/PI− (blue), Ann.V+/PI−(yellow), Ann.V+/PI+ (red), Ann.V−/PI+ (black). Error as within 5% (datanot shown). NC—normal condition, SS—serum starvation.

FIG. 29 shows that the absence of Akt1 does not impede 4E-dependentnuclear-cytoplasmic transport of reported sensitive RNA targets. (A) RNAquantification from parallel real time PCR experiments from MEF Akt1 wtand Akt1−/− derived cells show the relative fold increase (y-axis) ofthe cytoplasmic/nuclear ratio of NBS1, cyclin D1 (positive control for4E dependent mRNA export) and VEGF (negative control) mRNAs. Shown beloware controls for sample fractionation (U6 SnRNA—nuclear,tRNAlys—cytoplasmic). (B) Control experiment showing total levels ofNBS1, cyclin D1 or VEGF RNA, with western analysis shown belowindicating changes at the protein expression level. Bar representationis as indicated. Cytoplasmic/nuclear ratios represent relative folddifference SD normalized to vector control which was set to 1. Averagedvalues of all analyzed mRNAs were normalized to GAPDH mRNA values.

FIG. 30 shows that NBS1 expression is necessary for upregulation of theAkt1 pathway by 4E. (A) Western blot analysis of whole-cell extractsfrom siRNA treated MEF Akt wt derived cells; scram=scrambled control,siNBS1=extracts from cells treated with siRNA for NBS1. Proteinsdetected are as indicated. β-actin is shown as a protein loadingcontrol. (B) Quantification of viable cells from apoptosis assays(Ann.V−/PI−) of siNBS1 treated Akt1 wt derived cells (vector versus 4E).Error was within 10%. NC—normal condition, SS—serum starvation. (C)Visual confirmation of apoptosis of siNBS1 treated Akt1 wt derived cells(as indicated) from TUNEL experiments (panels: blue—DAPI (viable),red—apoptotic). Scale bar is set at 50 μm.

FIG. 31 shows that overexpression of the 4E inhibitor, PML, abrogatesthe 4E—Akt1 pathway, while the PML RING mutant does not inhibit these 4Edependent activities. (A) Western blot analysis of whole-cell extractsfrom stably transfected NIH3T3 cells over-expressing 4E wt/W73A and/orPML/RING. Proteins detected are as indicated. Note that the antibodyused for PML detection only binds to the exogenous PML. β-actin is shownas a protein loading control. (B) Parallel qPCR experiments showingrelative fold increase (y-axis) of the cytoplasmic/nuclear ratio of 4Etarget mRNAs from NIH3T3 derived cells. Bar representation is asindicated.

FIG. 32 shows that the 4E inhibitor, PML, relieves cells from4E-dependent apoptotic rescue through the RING domain of PML. (A) Bargraphs represent quantitative measurements of apoptosis using Annexin V(Ann.V) and propidium iodide (PI) staining of indicated NIH3T3 derivedcells. Bar color is as follows: Ann.V−/PI− (blue), Ann.V+/PI− (yellow),Ann.V+/PI+ (red), Ann.V−/PI+ (black). Error was within 5% (data notshown). NC—normal condition, SS—serum starvation. (B) Visualconfirmation of apoptosis from TUNEL experiments (panels: blue—DAPI,red—apoptotic). Scale bar is set at 50 μm.

FIG. 33 depicts comparison models summarizing how 4E is not onlydownstream of PI3K—AKT pathway (left), but can modulate this PI3K—Aktaxis through NBS1 (right). Further, several downstream targets of Akt(eg: cyclin A2, B1, D1, E; Mdm2, and c-Myc) are also targets for 4Eregulation at the mRNA transport level, giving rise to a putativefeedback loop. For simplicity, arrows indicate downstream effects (suchas phosphorylation), thus arrows do not necessarily indicate a singlestep process. Boxed in yellow are some of the known subset of mRNAssensitive to 4E transport activity that also play a role in the Aktpathway.

FIG. 34 depicts the components of the 4E regulon.

FIGS. 35 and 36 show that Akt phosphorylation is required for activationof Akt. Ribavirin inhibits Akt phosphorylation while Rapamycin increasesAkt phosphorylation.

FIG. 37 shows that Ribavirin blocks 4E mediated apoptotic rescue andRapamycin partially inhibits Ribavirin effect on 4E mediated apoptoticrescue.

FIG. 38 depicts the effect of Ribavirin on the protein levels of 4E,actin, NBS1, Cyclin D1 and ODC on FaDu cells were grown in culture andtreated with Ribavirin for 48 hours prior to preparation of proteinextracts and western blot analysis.

DETAILED DESCRIPTION

A. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

-   -   The terms “4E” and “eIF4e” are used herein interchangeably.

The term “4E activity” or “activity of 4E” includes any of thebiological effects of the 4E gene or protein, including but not limitedto elevated expression of 4E, elevated protein levels of 4E, 4E regulonactivity and/or activation of 4E regulon components, expression and/oractivity or level of components under control of the 4E regulon,elevated transport of selected messages (especially cyclin D1 andadditional messages as detailed in Examples 2 and 4) from the nucleus tothe cytoplasm, and phosphorylation state of 4E and levels of eIF4EBP1.

The term “4E regulon activity” or “4E regulon component activity” or“activity of a 4E regulon component” refers the activity of 4E as amediator of the 4E regulon and also includes 4E regulon activation,expression, transport and/or activity of the 4E regulon components.

The term “4E regulon component” refers to 4E, any of the components ofits regulon, and any modifier of the regulon such as HuR. The 4E regulonis illustrated in FIGS. 24 and 34. Exemplary 4E regulon elementsinclude: eIF4E (gi: 54873625); Cyclin D1 (gi: 77628152); NBS/Nibrin (gi:67189763); Pim-1 (gi: 31543400); Cyclin B1 (gi: 34304372); Cyclin A2(gi: 16950653); ODC (gi: 4505488); VEGF (gi: 71051577); Skp2 (gi:16306594, 16306593); Cyclin E1 (gi: 17318558); c-myc (gi: 71774082);FGF2 (gi: 153285460); MMP-9 (gi: 74272286); mdm2 (gi: 46488903);caspase-9 (gi: 14790123, 14790127); bcl2 (gi: 72198188, 72198345);Bcl/xL (gi: 20336334); Fbox1 (gi: 16306583); CGGbp1 (gi: 56550052);P54nrb/NONO.1 (gi: 34932413); Selenoprotein S (gi: 45439347); eIF4E-BP1(gi: 117938308); Akt1 (gi: 62241012, 62241010, 62241014); PI3K (gi:54792081, 212377724); GSK3B (gi: 21361339); HuR (gi: 38201713); andmTOR/FRAP1 (gi: 19924298). Preferred 4E regulon components (elements) tobe used in certain of the below-described methods are 4E, 4E-BP1,NBS/Nibrin, Pim-1, VEGF, Cyclin D1, Cyclin A2, ODC and HuR A “regulon”is a family of multiple mRNAs that are coordinately regulated in asequence specific fashion by one or more RNA binding proteins thatorchestrate and control their splicing, export, stability, localizationand/or translation.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms are art-recognized andrepresent methyl, ethyl, phenyl, trifluoromethanesulfonyl,nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,respectively. A more comprehensive list of the abbreviations utilized byorganic chemists of ordinary skill in the art appears in the first issueof each volume of the Journal of Organic Chemistry; this list istypically presented in a table entitled Standard List of Abbreviations.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “acylamino” is art-recognized and includes a moiety that may berepresented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedabove.

The term “administering” includes any method of delivery of a compoundof the present invention, including but not limited to, a pharmaceuticalcomposition or therapeutic agent, into a subject's system or to aparticular region in or on a subject. The phrases “systemicadministration,” “administered systemically,” “peripheraladministration” and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the patient'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration. “Parenteral administration” and“administered parenterally” means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The term “agonist”, as used herein, is meant to refer to an agent thatmimics or up-regulates (e.g., potentiates or supplements) thebioactivity of a protein. An agonist can be a wild-type protein orderivative thereof having at least one bioactivity of the wild-typeprotein. An agonist can also be a compound that upregulates expressionof a gene or which increases at least one bioactivity of a protein. Anagonist can also be a compound which increases the interaction of apolypeptide with another molecule, e.g., a target peptide or nucleicacid.

The term “aliphatic” is an art-recognized term and includes linear,branched, and cyclic alkanes, alkenes, or alkynes. In certainembodiments, aliphatic groups in the present invention are linear orbranched and have from 1 to about 20 carbon atoms.

The terms “alkenyl” and “alkynyl” are art-recognized, and includeunsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The terms “alkoxyl” or “alkoxy” are art-recognized and include an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R61, where m and R61 are described above.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure.

Moreover, the term “alkyl” (or “lower alkyl”) includes both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents mayinclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylsmay be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The definition of each expression, e.g. alkyl, m, n, etc., when itoccurs more than once in any structure, is intended to be independent ofits definition elsewhere in the same structure unless otherwiseindicated expressly or by the context.

The term “alkylthio” is art-recognized and includes an alkyl group, asdefined above, having a sulfur radical attached thereto. In certainembodiments, the “alkylthio” moiety is represented by one of —S-alkyl,—S-alkenyl, —S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 aredefined above. Representative alkylthio groups include methylthio, ethylthio, and the like.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The terms “amine” and “amino” are art-recognized and include bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In certain embodiments, only oneof R50 or R51 may be a carbonyl, e.g., R50, R51 and the nitrogentogether do not form an imide. In other embodiments, R50 and R51 (andoptionally R52) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “aralkyl” is art-recognized, and includes alkyl groupssubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to tencarbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

“Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g., suppresses or inhibits) at least one bioactivity ofa protein. An antagonist can be a compound which inhibits or decreasesthe interaction between a protein and another molecule, e.g., a targetpeptide or enzyme substrate. An antagonist can also be a compound thatdownregulates expression of a gene or which reduces the amount ofexpressed protein present.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments thereof which are also specifically reactive with avertebrate, e.g., mammalian, protein. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. Thus, the termincludes segments of proteolytically-cleaved or recombinantly-preparedportions of an antibody molecule that are capable of selectivelyreacting with a certain protein. Nonlimiting examples of suchproteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv,and single chain antibodies (scFv) containing a V[L] and/or V[H] domainjoined by a peptide linker. The scFv's may be covalently ornon-covalently linked to form antibodies having two or more bindingsites. The subject invention includes polyclonal, monoclonal, or otherpurified preparations of antibodies and recombinant antibodies.

The term “aryl” is art-recognized, and includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring may be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The term “binding” refers to an association, which may be a stableassociation, between two molecules, e.g., between a polypeptide of theinvention and a binding partner, due to, for example, electrostatic,hydrophobic, ionic and/or hydrogen-bond interactions under physiologicalconditions.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably, refer to an effector orantigenic function that is directly or indirectly performed by apolypeptide (whether in its native or denatured conformation), or by anysubsequence thereof. Biological activities include binding topolypeptides, binding to other proteins or molecules, activity as a DNAbinding protein, as a transcription regulator, ability to bind damagedDNA, phosphorylation state, etc. A bioactivity may be modulated bydirectly affecting the subject polypeptide. Alternatively, a bioactivitymay be altered by modulating the level of the polypeptide, such as bymodulating expression of the corresponding gene.

The term “biological sample”, or “sample” as used herein, refers to asample obtained from an organism or from components (e.g., cells) of anorganism. The sample may be of any biological tissue or fluid.Frequently the sample will be a “clinical sample” which is a samplederived from a patient. Such samples include, but are not limited to,sputum, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes.

The term “cancer” refers in general to any malignant neoplasm orspontaneous growth or proliferation of cells. The term as used hereinencompasses both fully developed malignant neoplasms, as well aspremalignant lesions. A subject having “cancer”, for example, may have atumor or a white blood cell proliferation such as leukemia. In certainembodiments, a subject having cancer is a subject having a tumor, suchas a solid tumor. Cancers include but are not limited to non small celllung cancer (NSCLC), testicular cancer, lung cancer, ovarian cancer,uterine cancer, cervical cancer, pancreatic cancer, colorectal cancer(CRC), breast cancer, prostate cancer, gastric cancer, skin cancer,stomach cancer, esophageal cancer, bladder cancer, thyroid cancer,parathyroid cancer, brain cancer, biliary cancer, rhabdomyosarcoma, headand neck cancer, tuberous sclerosis and blood cancers including but notlimited to non-Hodgkin's lymphoma (NHL), acute myelogenous leukemia(AML) and blast crisis of chronic myelogenous leukemia (bc-CML).

The term “carbocycle” is art-recognized and includes an aromatic ornon-aromatic ring in which each atom of the ring is carbon. Thefollowing art-recognized terms have the following meanings: “nitro”means —NO₂; the term “halogen” designates —F, —Cl, —Br or —I; the term“sulfhydryl” means —SH; the term “hydroxyl” means —OH; and the term“sulfonyl” means —SO₂ ⁻.

The term “carbonyl” is art-recognized and includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61, or apharmaceutically acceptable salt. R56 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R61, where m and R61 are defined above. WhereX50 is an oxygen and R55 or R56 is not hydrogen, the Formula representsan “ester”. Where X50 is an oxygen, and R55 is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR55 is a hydrogen, the Formula represents a “carboxylic acid”. Where X50is an oxygen, and R56 is hydrogen, the Formula represents a “formate”.In general, where the oxygen atom of the above Formula is replaced bysulfur, the Formula represents a “thiocarbonyl” group. Where X50 is asulfur and R55 or R56 is not hydrogen, the Formula represents a“thioester.” Where X50 is a sulfur and R55 is hydrogen, the Formularepresents a “thiocarboxylic acid.” Where X50 is a sulfur and R56 ishydrogen, the Formula represents a “thioformate.” On the other hand,where X50 is a bond, and R55 is not hydrogen, the above Formularepresents a “ketone” group. Where X50 is a bond, and R55 is hydrogen,the above Formula represents an “aldehyde” group.

A “combinatorial library” or “library” is a plurality of compounds,which may be termed “members,” synthesized or otherwise prepared fromone or more starting materials by employing either the same or differentreactants or reaction conditions at each reaction in the library. Ingeneral, the members of any library show at least some structuraldiversity, which often results in chemical diversity. A library may haveanywhere from two different members to about 10⁸ members or more. Incertain embodiments, libraries of the present invention have more thanabout 12, 50 and 90 members. In certain embodiments of the presentinvention, the starting materials and certain of the reactants are thesame, and chemical diversity in such libraries is achieved by varying atleast one of the reactants or reaction conditions during the preparationof the library. Combinatorial libraries of the present invention may beprepared in solution or on the solid phase.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “diagnosing” refers to determining the presence of a disease ina patient.

A “disease wherein 4E regulon activity is dysfunctional” refers to anycondition in a subject where the expression level of, activity of,amounts of, or phosphorylation states of the 4E regulon componentsdiffer statistically significantly from those observed in a nondiseasedsubject. Exemplary diseases wherein 4E regulon activity is dysfunctionalinclude cancer, proliferation disorders, ischemia reperfusion andhypertrophy.

The term “effective amount” refers to that amount of a compound,material, or composition comprising a compound of the present inventionwhich is sufficient to effect a desired result, including, but notlimited to, for example, reducing tumor volume either in vitro or invivo. An effective amount of a pharmaceutical composition of the presentinvention is an amount of the pharmaceutical composition that issufficient to effect a desired clinical result, including but notlimited to, for example, ameliorating, stabilizing, preventing ordelaying the development of cancer in a patient. In either case, aneffective amount of the compounds of the present invention can beadministered in one or more administrations. Detection and measurementof these above indicators are known to those of skill in the art,including, but not limited for example, reduction in tumor burden,inhibition of tumor size, reduction in proliferation of secondarytumors, expression of genes in tumor tissue, presence of biomarkers,lymph node involvement, histologic grade, and nuclear grade.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, March, Advanced Organic Chemistry 251-59, McGraw Hill BookCompany, New York, (1977). The Hammett constant values are generallynegative for electron donating groups (σ(P)=−0.66 for NH₂) and positivefor electron withdrawing groups (σ(P)=0.78 for a nitro group), σ(P)indicating para substitution. Exemplary electron-withdrawing groupsinclude nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride,and the like. Exemplary electron-donating groups include amino, methoxy,and the like.

“Gene” or “recombinant gene” refers to a nucleic acid moleculecomprising an open reading frame and including at least one exon and(optionally) an intron sequence. “Intron” refers to a DNA sequencepresent in a given gene which is spliced out during mRNA maturation.

By “gene product” it is meant a molecule that is produced as a result oftranscription of a gene. Gene products include RNA molecules transcribedfrom a gene, as well as proteins translated from such transcripts.

The term “heteroatom” is art-recognized, and includes an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium, andalternatively oxygen, nitrogen or sulfur.

The terms “heterocyclyl” and “heterocyclic group” are art-recognized,and include 3- to about 10-membered ring structures, such as 3- to about7-membered rings, whose ring structures include one to four heteroatoms.Heterocycles may also be polycycles. Heterocyclyl groups include, forexample, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene,xanthene, phenoxathin, pyrrole, imidazole, pyrazole, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones, and the like. Theheterocyclic ring may be substituted at one or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “hydrocarbon” is art-recognized and includes all permissiblecompounds having at least one hydrogen and one carbon atom. For example,permissible hydrocarbons include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticorganic compounds that may be substituted or unsubstituted.

The phrase “hydroxyl-protecting group” is art-recognized and includesthose groups intended to protect a hydroxyl group against undesirablereactions during synthetic procedures and includes, for example, benzylor other suitable esters or ethers groups known in the art.

The term “hypertrophy” refers in general to any abnormal enlargement ofa body part or organ.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “ischemia reperfusion” refers in general to refers to damage totissue caused when blood supply returns to the tissue after a period ofischemia. “Ischemia” refers to a low oxygen state usually due toobstruction of the arterial blood supply or inadequate blood flowleading to hypoxia in the tissue.

The terms “label” or “labeled” refer to incorporation or attachment,optionally covalently or non-covalently, of a detectable marker into amolecule, such as a polypeptide and especially an antibody. Variousmethods of labeling polypeptides are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes, fluorescent labels, heavy atoms, enzymaticlabels or reporter genes, chemiluminescent groups, biotinyl groups,predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). Examples and use ofsuch labels are described in more detail below. In some embodiments,labels are attached by spacer arms of various lengths to reducepotential steric hindrance. Particular examples of labels which may beused under the invention include fluorescein, rhodamine, dansyl,umbelliferone, Texas red, luminol, NADPH, alpha-beta-galactosidase andhorseradish peroxidase.

The “level of expression of a gene in a cell” or “gene expression level”refers to the level of mRNA, as well as pre-mRNA nascent transcript(s),transcript processing intermediates, mature mRNA(s) and degradationproducts, encoded by the gene in the cell.

The term “modulation”, when used in reference to a functional propertyor biological activity or process (e.g., enzyme activity or receptorbinding), refers to the capacity to either up regulate (e.g., activateor stimulate), down regulate (e.g., inhibit or suppress) or otherwisechange a quality of such property, activity or process. In certaininstances, such regulation may be contingent on the occurrence of aspecific event, such as activation of a signal transduction pathway,and/or may be manifest only in particular cell types.

The term “modulator” refers to a polypeptide, nucleic acid,macromolecule, complex, molecule, small molecule, compound, species orthe like (naturally-occurring or non-naturally-occurring), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal cells or tissues, that may be capable of causing modulation.Modulators may be evaluated for potential activity as inhibitors oractivators (directly or indirectly) of a functional property, biologicalactivity or process, or combination of them, (e.g., agonist, partialantagonist, partial agonist, inverse agonist, antagonist, anti-microbialagents, inhibitors of microbial infection or proliferation, and thelike) by inclusion in assays. In such assays, many modulators may bescreened at one time. The activity of a modulator may be known, unknownor partially known.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides. ESTs, chromosomes,cDNAs, mRNAs, and rRNAs are representative examples of molecules thatmay be referred to as nucleic acids.

The terms ortho, meta and sara are art-recognized and apply to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

A “patient” or “subject” or “host” refers to either a human or non-humananimal.

The term “pharmaceutical delivery device” refers to any device that maybe used to administer a therapeutic agent or agents to a subject.Non-limiting examples of pharmaceutical delivery devices includehypodermic syringes, multichamber syringes, stents, catheters,transcutaneous patches, microneedles, microabraders, and implantablecontrolled release devices. In one embodiment, the term “pharmaceuticaldelivery device” refers to a dual-chambered syringe capable of mixingtwo compounds prior to injection.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

“Pharmaceutically-acceptable salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds.

The term “phosphonamidite” is art-recognized and includes moietiesrepresented by the general formulas:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents alower alkyl or an aryl.

The term “phosphoryl” is art-recognized and includes moietiesrepresented by the general formula:

wherein Q50 represents S or O, and R59 represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q50 and R59, each independently, are defined above, and Q51represents O, S or N. When Q50 is S, the phosphoryl moiety is a“phosphorothioate”.

The term “phosphoramidite” is art-recognized and includes moietiesrepresented by the general formulas:

wherein Q51, R50, R51 and R59 are as defined above.

The terms “polycyclyl” and “polycyclic group” are art-recognized, andinclude structures with two or more rings (e.g., cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which twoor more carbons are common to two adjoining rings, e.g., the rings are“fused rings”. Rings that are joined through non-adjacent atoms, e.g.,three or more atoms are common to both rings, are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “prognosing” refers to determining the probable outcome of anattack of disease or the prospect as to recovery from a disease asindicated by the nature and symptoms of the case.

The phrase “protecting group” is art-recognized and includes temporarysubstituents that protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed. Greene et al., ProtectiveGroups in Organic Synthesis 2^(nd) ed., Wiley, New York, (1991).

“Protein”, “polypeptide” and “peptide” are used interchangeably hereinwhen referring to a gene product, e.g., as may be encoded by a codingsequence. By “gene product” it is meant a molecule that is produced as aresult of transcription of a gene. Gene products include RNA moleculestranscribed from a gene, as well as proteins translated from suchtranscripts.

A “regulon” is a collection of genes under regulation by the sameregulatory protein. The “4E regulon” is the collection of genes (“4Eregulon components”) under regulation by 4E, as described in theExamples and shown in FIG. 24, and thus includes 4E as a component.

“Small molecule” is an art-recognized term. In certain embodiments, thisterm refers to a molecule which has a molecular weight of less thanabout 2000 amu, or less than about 1000 amu, and even less than about500 amu.

The term “staging” refers to determining the degree to which a diseasehas progressed in a subject.

It will be understood that the terms “substitution” and “substitutedwith” are art-recognized and include the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, e.g., which does not spontaneously undergo transformation suchas by rearrangement, cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The term “sulfonate” is art-recognized and includes a moiety that may berepresented by the general formula:

in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art-recognized and includes a moiety that may berepresented by the general formula:

in which R57 is as defined above.

The term “sulfonamido” is art-recognized and includes a moiety that maybe represented by the general formula:

in which R50 and R56 are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that may berepresented by the general formula:

in which R50 and R51 are as defined above.

The term “sulfonyl” is art-recognized and includes a moiety that may berepresented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and includes a moiety that may berepresented by the general formula:

in which R58 is defined above.

“Therapeutic agent” or “therapeutic” refers to an agent capable ofhaving a desired biological effect on a host. Chemotherapeutic andgenotoxic agents are examples of therapeutic agents that are generallyknown to be chemical in origin, as opposed to biological, or cause atherapeutic effect by a particular mechanism of action, respectively.Examples of therapeutic agents of biological origin include growthfactors, hormones, and cytokines. A variety of therapeutic agents areknown in the art and may be identified by their effects. Certaintherapeutic agents are capable of regulating red cell proliferation anddifferentiation. Examples include chemotherapeutic nucleotides, drugs,hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g.,antisense oligonucleotides that bind to a target nucleic acid sequence(e.g., mRNA sequence)), peptides, and peptidomimetics.

The term “therapeutically effective amount” refers to that amount of amodulator, drug or other molecule which is sufficient to effecttreatment when administered to a subject in need of such treatment. Thetherapeutically effective amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of any condition or disease.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

B. Small Molecule Compositions that Regulate 4E Activity, CellProliferation and Cancer

In parallel studies, we examined the potential role of guanosineribonucleoside analog and anti-viral agent Ribavirin (Sidwell, R. W., etal. (1972). Science 177:705-6.) in the regulation of 4E activity withinthe cell. Ribavirin is currently used for the treatment of infectionswith Lassa Fever virus, respiratory syncytial virus, hepatitis C virusand sever acute respiratory syndrome coronavirus. Mechanistically, ithas been demonstrated recently that Ribavirin can misincorporated intomRNA by viral RNA-dependent RNA polymerases because of its chemicalsimilarity to guanosine, and this outcome leads to the lethalmutagenesis of genomes of polio and HCV. Ribavirin triphosphate (RTP)binds the HCV polymerase with an observed dissociation constant of 0.58mM which is consistent with the micromolar concentrations required toachieve a therapetuci effect against HCV clinically. By contrast to itseffects at millimolar concentrations, Ribavirin inhibits the growth ofhuman lymphocytes at micromolar levels, yet with no clear mechanism ofaction reported.

We determined that Ribavirin binds directly to 4E with low micromolaraffinity in vitro; that this interaction occurs at concentrations500-fold lower than those required for previously demonstratedactivities of Ribavirin, that Ribavirin efficiently competes with 4Ebinding of the m7G mRNA cap in vitro and in cells at high nanomolar tolow micromolar concentrations; that at these concentrations Ribavirinspecifically mediates the disruption of 4E:m7G functions in thetransport and translation of 4E-regulated genes at low micromolarconcentrations in cells; that administration of high nanomolar to lowmicromolar concentrations of Ribavirin specifically down-regulatesoncogenic protein production, induces cell cycle arrest therebysuppressing overall 4E activity in vitro and in vivo.

We have found that Ribavirin and new chemical entities/derivativesthereof possess the capacity to selectively inhibit the biological,proliferative and oncogenic properties of elevated 4E activity withincells, tissues and tumors. Unexpectedly, this targeted inhibition ofelevated 4E activity does not impact other biological processes withinthe cells.

In particular, we have found that Ribavirin and new chemicalentities/derivatives thereof possess the capacity to inhibit 4E regulonactivity. Ribavirin has been observed to impede 4E regulon activity andreduce intracellular levels of 4E regulon components.

Accordingly, provided are small molecule compositions comprising smallmolecules of the following formula:

wherein:

R1 may be a linear or branched alkyl, alkenyl, hydrogen, alkynyl, andthe like. Preferably, R1 is —H, —CH₃, or CH₂CH₃.

R2 may be an amine (primary, secondary, and tertiary, linear orbranched), an aromatic amine, an amino group or an amido group.Preferably, R2 is —NH₂, —NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₂)₂—NHCH₂CH₂CH₂OH, —NHCH₂CH₂CH(OH)CH₃, and —NHCH(CH₂OH)CH₃.

R3 may be oxygen or sulfur; and

R4 may be a hydroxyl group, a phosphate group, siloxane, carbonate,carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,sulfonate, sulfonamide, thioformacetal, formacetal, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino. The phosphategroup, when present, may be optionally attached to at least one base,and is of the formula:

Ribovirin and its analogs are physical mimics of 7-methyl guanosine(m⁷G). 7-methyl guanosine (m⁷G) and analogs thereof (depicted in FormulaII below) are also expected to inhibit 4E activity, in particular 4Eregulon activity:

wherein:

R1 and R2 each independently may be a linear or branched alkyl, alkenyl,hydrogen, alkynyl, and the like. Preferably, R1 is —H, —CH₃, or CH₂CH₃.Preferably, R2 is —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CH₂CH₂OH,—CH₂CH₂CH(OH)CH₃, or —CH(CH₂OH)CH₃;

R3 may be oxygen or sulfur;

R4 may be a hydroxyl group, a phosphate group, siloxane, carbonate,carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,sulfonate, sulfonamide, thioformacetal, formacetal, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino. The phosphategroup, when present, may be optionally attached to at least one base,and is of the formula:

R5 may be an amine (primary, secondary, and tertiary, linear orbranched), an aromatic amine, an amino group or an amido group.

Physically ribavirin is similar to the sugar D-ribose from which it isderived. It is freely soluble in water, and is re-crystallized as finesilvery needles from boiling methanol. It is only sparingly soluble inanhydrous ethanol. Classically ribavirin is prepared from naturalD-ribose by blocking the 2′, 3′ and 5′ OH groups with benzyl groups,then derivatizing the 1′ OH with an acetyl group which acts as asuitable leaving group upon nucleophilic attack. The ribose 1′ carbonattack is accomplished with 1,2,4 triazole-3-carboxymethyl ester, whichdirectly attaches the 1′ nitrogen of the triazole to the 1′ carbon ofthe ribose, in the proper 1-β-D isomeric position. The bulky benzylgroups hinder attack at the other sugar carbons. Following purificationof this intermediate, treatment with ammonia in methanolic conditionsthen simultaneously deblocks the ribose hydroxyls, and converts thetriazole carboxymethyl ester to the carboxamide. Following this step,ribavirin may be recovered in good quantity by cooling andcrystallization.

Ribavirin is possibly best viewed as a ribosyl purine analogue with anincomplete purine 6-membered ring. This structural resemblancehistorically prompted replacement of the 2′ nitrogen of the triazolewith a carbon (which becomes the 5′ carbon in an imidazole), in anattempt to partly “fill out” the second ring—but to no great effect.Such 5′ imidazole riboside derivatives show antiviral activity with 5′hydrogen or halide, but the larger the substituent, the smaller theactivity, and all proved less active than ribavirin (Harris, S. &Robins, R. K. (1980). Ribavirin: structure and antiviral activityrelationships. In Ribavirin: A Broad Spectrum Antiviral Agent (Smith, R.A. & Kirkpatrick, W., Eds), pp. 1-21. Academic Press, New York, N.Y.,USA). Note that two natural products were already known with thisimidazole riboside structure: substitution at the 5′ carbon with OHresults in pyrazomycin/pyrazofurin, an antibiotic with antiviralproperties but unacceptable toxicity, and replacement with an aminogroup results in the natural purine synthetic precursor5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which hasonly modest antiviral properties.

Derivatization of the triazole 5′ carbon, or replacement of it with anitrogen (i.e., the 1,2,4,5 tetrazole 3-carboxamide) also results insubstantial loss of activity, as does alkyl derivatization of the 3′carboxamide nitrogen.

The 2′ deoxyribose version of ribavirin (the DNA nucleoside analogue) isnot active as an antiviral, suggesting strongly that ribavirin requiresRNA-dependent enzymes for its antiviral activity.

Antiviral activity is retained for acetate and phosphate derivation ofthe ribose hydroxyls, including the triphosphate and 3′, 5′ cyclicphosphates, but these compounds are no more active than the parentmolecule, reflecting the high efficiency of esterase and kinase activityin the body.

Modifications of Formula I and Formula II can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., an RRMS, as described herein;

(iii) wholesale replacement of the phosphate with “dephospho” linkers;

(iv) replacement or modification of the ribose-phosphate backbone(bracket II);

(v) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic but acid rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e. Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X═O and Y═N in the above figures.

Certain compositions of the present invention may exist in particulargeometric or stereoisomeric forms. In addition, certain compositions ofthe present invention may also be optically active. The presentinvention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Additional specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms (i.e., X and Yabove). However, the phosphate group can be modified by replacing one ofthe oxygens with a different substituent. One result of thismodification to RNA phosphate backbones can be increased resistance ofthe oligoribonucleotide to nucleolytic breakdown. Thus while not wishingto be bound by theory, it can be desirable in some embodiments tointroduce alterations which result in either an uncharged linker or acharged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. Unlike the situation where only one of X or Y isaltered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of oligoribonucleotides diastereomers.Diastereomer formation can result in a preparation in which theindividual diastereomers exhibit varying resistance to nucleases.Further, the hybridization affinity of RNA containing chiral phosphategroups can be lower relative to the corresponding unmodified RNAspecies. Thus, while not wishing to be bound by theory, modifications toboth X and Y which eliminate the chiral center, e.g. phosphorodithioateformation, may be desirable in that they cannot produce diastereomermixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linkingoxygen (i.e., W or Z) with nitrogen (bridged phosphoroamidates), sulfur(bridged phosphorothioates) and carbon (bridged methylenephosphonates).The replacement can occur at a terminal oxygen (position W (3′) orposition Z (5′). Replacement of W with carbon or Z with nitrogen ispreferred.

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. Nos. 5,256,775 or 5,366,878. The preparation of phosphotriesteroligoribonucleotides is described in U.S. Pat. No. 5,023,243. Thepreparation of borano phosphate oligoribonucleotide is described in U.S.Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-aminophosphoramidate oligoribonucleotides is described in U.S. Pat. No.5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides isdescribed in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.Preparation of sulfur bridged nucleotides is described in Sproat et al.Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. TetrahedronLett. 1989, 30, 4693.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. e.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modification.

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group

The phosphate group, can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula I above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. Examples include 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity.

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be with solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter3,2′-O—Methyloligoribonucleotide-s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Jyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Jyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

The most successful ribavirin derivative to date is the 3-carboxamidinederivative of the parent 3-carboxamide(1-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2,4-triazole-3-carboximidamide),first reported in 1973 by Witkowski, and now generally called viramidine(also “Ribamidine”). Considerations in designing prodrugs of ribavirinuseful in the methods and compositions provided herein are discussed inWu, et al. Journal of Antimicrobial Chemotherapy (2003) 52, 543-546.

Other ribavirin prodrugs and/or analogues/derivatives specificallycontemplated for use in the methods and compositions provided hereinare: methimazole, carbimazole, metronidazole, selanazofurin,showdomycin, pyrazomycin and ICN 3297, and analogs and derivativesthereof.

A number of techniques can be used to screen, identify, select anddesign chemical entities capable of inhibiting 4E activity, based on thestructures described above. The term “chemical entity,” as used herein,refers to chemical compounds, complexes of two or more chemicalcompounds, and fragments of such compounds or complexes. In certaininstances, it is desirable to use chemical entities exhibiting a widerange of structural and functional diversity, such as compoundsexhibiting different shapes (e.g., flat aromatic rings(s), puckeredaliphatic rings(s), straight and branched chain aliphatics with single,double, or triple bonds) and diverse functional groups (e.g., carboxylicacids, esters, ethers, amines, aldehydes, ketones, and variousheterocyclic rings).

In one aspect, the method of drug design generally includescomputationally evaluating the potential of a selected chemical entityto associate with 4E (or portions thereof). For example, this method mayinclude the steps of (a) employing computational means to perform afitting operation between the selected chemical entity and a druggableregion of 4E; and (b) analyzing the results of said fitting operation toquantify the association between the chemical entity and 4E.

A chemical entity may be examined either through visual inspection orthrough the use of computer modeling using a docking program such asGRAM, DOCK, or AUTODOCK (Dunbrack et al., Folding & Design, 2:27-42(1997)). This procedure can include computer fitting of chemicalentities to a target to ascertain how well the shape and the chemicalstructure of each chemical entity will complement or interfere with thestructure of the subject polypeptide (Bugg et al., Scientific American,December: 92-98 (1993); West et al., TIPS, 16:67-74 (1995)). Computerprograms may also be employed to estimate the attraction, repulsion, andsteric hindrance of the chemical entity to a druggable region, forexample. Generally, the tighter the fit (e.g., the lower the sterichindrance, and/or the greater the attractive force) the more potent thechemical entity will be because these properties are consistent with atighter binding constant. Furthermore, the more specificity in thedesign of a chemical entity the more likely that the chemical entitywill not interfere with related proteins, which may minimize potentialside-effects due to unwanted interactions.

A variety of computational methods for molecular design, in which thesteric and electronic properties of druggable regions are used to guidethe design of chemical entities, are known: Cohen et al. (1990) J. Med.Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol. 161: 269-288;DesJarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) Spec.Publ., Roy. Soc. Chem. 78: 182-196; Goodford et al. (1985) J. Med. Cam.28: 849-857; and DesJarlais et al. J. Med. Cam. 29: 2149-2153. Directedmethods generally fall into two categories: (1) design by analogy inwhich 3-D structures of known chemical entities (such as from acrystallographic database) are docked to the druggable region and scoredfor goodness-of-fit; and (2) de novo design, in which the chemicalentity is constructed piece-wise in the druggable region. The chemicalentity may be screened as part of a library or a data base of molecules.Data bases which may be used include ACD (Molecular Designs Limited),NCI (National Cancer Institute), CCDC (Cambridge Crystallographic DataCenter), CAST (Chemical Abstract Service), Derwent (Derwent InformationLimited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (AldrichChemical Company), DOCK (University of California in San Francisco), andthe Directory of Natural Products (Chapman & Hall). Computer programssuch as CONCORD (Tripos Associates) or DB-Converter (MolecularSimulations Limited) can be used to convert a data set represented intwo dimensions to one represented in three dimensions.

Chemical entities may be tested for their capacity to fit spatially witha druggable region or other portion of 4E. As used herein, the term“fits spatially” means that the three-dimensional structure of thechemical entity is accommodated geometrically by a druggable region. Afavorable geometric fit occurs when the surface area of the chemicalentity is in close proximity with the surface area of the druggableregion without forming unfavorable interactions. A favorablecomplementary interaction occurs where the chemical entity interacts byhydrophobic, aromatic, ionic, dipolar, or hydrogen donating andaccepting forces. Unfavorable interactions may be steric hindrancebetween atoms in the chemical entity and atoms in the druggable region.

If a model of the present invention is a computer model, the chemicalentities may be positioned in a druggable region through computationaldocking. If, on the other hand, the model of the present invention is astructural model, the chemical entities may be positioned in thedruggable region by, for example, manual docking. As used herein theterm “docking” refers to a process of placing a chemical entity in closeproximity with a druggable region, or a process of finding low energyconformations of a chemical entity/druggable region complex.

In an illustrative embodiment, the design of potential modulator beginsfrom the general perspective of shape complimentary for the druggableregion of a polypeptide of the invention, and a search algorithm isemployed which is capable of scanning a database of small molecules ofknown three-dimensional structure for chemical entities which fitgeometrically with the target druggable region. Most algorithms of thistype provide a method for finding a wide assortment of chemical entitiesthat are complementary to the shape of a druggable region of the subjectpolypeptide. Each of a set of chemical entities from a particulardata-base, such as the Cambridge Crystallographic Data Bank (CCDB)(Allen et al. (1973) J. Chem. Doc. 13: 119), is individually docked tothe druggable region of a polypeptide of the invention in a number ofgeometrically permissible orientations with use of a docking algorithm.In certain embodiments, a set of computer algorithms called DOCK, can beused to characterize the shape of invaginations and grooves that formthe active sites and recognition surfaces of the druggable region (Kuntzet al. (1982) J. Mol. Biol. 161: 269-288). The program can also search adatabase of small molecules for templates whose shapes are complementaryto particular binding sites of a polypeptide of the invention(DesJarlais et al. (1988) J Med Chem 31: 722-729).

The orientations are evaluated for goodness-of-fit and the best are keptfor further examination using molecular mechanics programs, such asAMBER or CHARMM. Such algorithms have previously proven successful infinding a variety of chemical entities that are complementary in shapeto a druggable region.

Goodford (1985, J Med Chem 28:849-857) and Boobbyer et al. (1989, J MedChem 32:1083-1094) have produced a computer program (GRID) which seeksto determine regions of high affinity for different chemical groups(termed probes) of the druggable region. GRID hence provides a tool forsuggesting modifications to known chemical entities that might enhancebinding. It may be anticipated that some of the sites discerned by GRIDas regions of high affinity correspond to “pharmacophoric patterns”determined inferentially from a series of known ligands. As used herein,a “pharmacophoric pattern” is a geometric arrangement of features ofchemical entities that is believed to be important for binding. Attemptshave been made to use pharmacophoric patterns as a search screen fornovel ligands (Jakes et al. (1987) J Mol Graph 5:41-48; Brint et al.(1987) J Mol Graph 5:49-56; Jakes et al. (1986) J Mol Graph 4: 12-20).

Yet a further embodiment of the present invention utilizes a computeralgorithm such as CLIX which searches such databases as CCDB forchemical entities which can be oriented with the druggable region in away that is both sterically acceptable and has a high likelihood ofachieving favorable chemical interactions between the chemical entityand the surrounding amino acid residues. The method is based oncharacterizing the region in terms of an ensemble of favorable bindingpositions for different chemical groups and then searching fororientations of the chemical entities that cause maximum spatialcoincidence of individual candidate chemical groups with members of theensemble. The algorithmic details of CLIX is described in Lawrence etal. (1992) Proteins 12:31-41.

In this way, the efficiency with which a chemical entity may bind to orinterfere with a druggable region may be tested and optimized bycomputational evaluation. For example, for a favorable association witha druggable region, a chemical entity must preferably demonstrate arelatively small difference in energy between its bound and fine states(i.e., a small deformation energy of binding). Thus, certain, moredesirable chemical entities will be designed with a deformation energyof binding of not greater than about 10 kcal/mole, and more preferably,not greater than 7 kcal/mole. Chemical entities may interact with adruggable region in more than one conformation that is similar inoverall binding energy. In those cases, the deformation energy ofbinding is taken to be the difference between the energy of the freeentity and the average energy of the conformations observed when thechemical entity binds to the target.

In this way, the present invention provides computer-assisted methodsfor identifying or designing a potential modulator of the activity of 4Eincluding: supplying a computer modeling application with a set ofstructure coordinates of a molecule or complex, the molecule or complexincluding at least a portion of a druggable region from 4E; supplyingthe computer modeling application with a set of structure coordinates ofa chemical entity; and determining whether the chemical entity isexpected to bind to the molecule or complex, wherein binding to themolecule or complex is indicative of potential modulation of theactivity of 4E.

In another aspect, the present invention provides a computer-assistedmethod for identifying or designing a potential modulator to 4E,supplying a computer modeling application with a set of structurecoordinates of a molecule or complex, the molecule or complex includingat least a portion of a druggable region of 4E; supplying the computermodeling application with a set of structure coordinates for a chemicalentity; evaluating the potential binding interactions between thechemical entity and active site of the molecule or molecular complex;structurally modifying the chemical entity to yield a set of structurecoordinates for a modified chemical entity, and determining whether themodified chemical entity is expected to bind to the molecule or complex,wherein binding to the molecule or complex is indicative of potentialmodulation of 4E.

An exemplary set of structural coordinates, the cap-free structure of4E, for use in the methods is described in Volpon, et al. (2006) EMBO J.25(21):5138-49. Epub 2006 Oct. 12. The activity of the eukaryotictranslation initiation factor 4E is modulated through conformationalresponse to its ligands. For example, eIF4G and 4E-BPs modulate capaffinity, and thus physiological activity of 4E, by binding a sitedistal to the m7G cap-binding site. Further, cap binding substantiallymodulates 4E's affinity for eIF4G and the 4E-BPs. Up to the date ofVolpon, et al, only cap-bound 4E structures were reported. In theabsence of structural information on the apo-form, the molecularunderpinnings of this conformational response mechanism cannot beestablished. The cap-free 4E structure shows structural differences inthe cap-binding site and dorsal surface relative to cap-eIF4E. Analysisof structure and dynamics of apo-eIF4E, and changes observed upon ligandbinding, reveal a molecular basis for eIF4E's conformational response tothese ligands. In particular, alterations in the S4-H4 loop, distal toeither the cap or eIF4G binding sites, appear key to modulating theseeffects. Mutation in this loop mimics these effects.

Cap-bound 4E structures, such as those described in Marcotrigiano, J.,et al. (1997) Cell, 89:951-961, Tomoo, K., et al. (2005) Biochim BiophysActa, 1753:191-208, Tomoo, K., et al. (2002) Biochem J. 362:539-544 andNiedzwiecka, A., et al. (2002) J Mol Biol, 319:615-635, may also be usedin the methods described herein.

In other embodiments, a potential modulator can be obtained by screeninga library. A potential modulator selected in this manner could be thenbe systematically modified by computer modeling programs and/or bysynthetic methods until one or more promising potential drugs areidentified. Such analysis has been shown to be effective in thedevelopment of HIV protease inhibitors (Lam et al., Science 263:380-384(1994); Wlodawer et al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt,Perspectives in Drug Discovery and Design 1:23-48 (1993); Erickson,Perspectives in Drug Discovery and Design 1: 109-128 (1993)).Alternatively a potential modulator may be selected from a library ofchemicals such as those that can be licensed from third parties, such aschemical and pharmaceutical companies. A third alternative is tosynthesize the potential modulator de novo.

Once a potential modulator is identified, it can then be tested in anystandard assay for 4E activity or 4E expression and protein levels, or4E regulon component expression and protein levels, including in highthroughput assays. Further refinements to the structure of the modulatorwill generally be necessary and can be made by the successive iterationsof any and/or all of the steps provided by the particular screeningassay. These studies may be performed in conjunction with biochemicalassays.

In any of the embodiments, the candidate compounds may be selected froma library of compounds. These libraries may be generated usingcombinatorial synthetic methods. The candidate compounds may beselected, for example, from the following classes of compounds:Ribavirin or ribavirin analogs, antisense nucleic acids, RNAi, smallmolecules, polypeptides, proteins, including antibodies,peptidomimetics, or nucleic acid analogs.

Specific, exemplary assays for 4E activity are described in theExemplification below. However, any method for determining the effect ofat least one candidate compound on 4E activity may be used. In certainembodiments, combinations of compounds or biologics may be screened fortheir effect on 4E activity to identify potential co-therapeutics orcombination therapies. For example, Ribavirin or analogs or prodrugsthereof may be screened along with interferon, GMCSF, GCSF, IL-12, IL-2,compounds that inhibit or down-regulated tyrosine kinase activity, etc.In addition to measurement of cell proliferation, cell division, and/orgene expression as noted

4E polypeptide may be used to assess the activity of small molecules andother modulators in in vitro assays. In one embodiment of such an assay,agents are identified which modulate the biological activity of 4E,4E-protein interaction of interest or 4E complex, such as an enzymaticactivity, binding to other cellular components, cellularcompartmentalization, signal transduction, and the like. In certainembodiments, the test agent is a small organic molecule. Analysis of theactivity and/or expression levels of the components of the 4E regulon,e.g., in a cell line expressing 4E and possessing the regulon, may alsobe used to assess the effect of a modulator on 4E activity.

Assays may employ kinetic or thermodynamic methodology using a widevariety of techniques including, but not limited to, microcalorimetry,circular dichroism, capillary zone electrophoresis, nuclear magneticresonance spectroscopy, fluorescence spectroscopy, and combinationsthereof.

Further provided are methods of screening compounds to identify thosewhich modulate 4E activity. The method of screening may involvehigh-throughput techniques. For example, to screen for modulators, asynthetic reaction mix, a cellular compartment, such as a membrane, cellenvelope or cell wall, or a preparation of any thereof, comprising 4Eand a labeled substrate or ligand of such polypeptide is incubated inthe absence or the presence of a candidate molecule that may be amodulator of 4E activity. The ability of the candidate molecule tomodulate 4E activity is reflected in decreased binding of the labeledligand or decreased production of product from such substrate. Detectionof the rate or level of production of product from substrate may beenhanced by using a reporter system. Reporter systems that may be usefulin this regard include but are not limited to colorimetric labeledsubstrate converted into product, a reporter gene that is responsive tochanges in 4E activity, and binding assays known in the art.

Another example of an assay for a modulator of 4E activity is acompetitive assay that combines 4E and a potential modulator withmolecules that bind to 4E, recombinant molecules that bind to 4E,natural substrates or ligands, or substrate or ligand mimetics, underappropriate conditions for a competitive inhibition assay. The 4E can belabeled, such as by radioactivity or a colorimetric compound, such thatthe number of molecules of 4E bound to a binding molecule or convertedto product can be determined accurately to assess the effectiveness ofthe potential modulator.

A number of methods for identifying a molecule which modulates 4Eactivity. For example, in one such method, a subject polypeptide iscontacted with a test compound, and the activity of the subjectpolypeptide in the presence of the test compound is determined, whereina change in the activity of the subject polypeptide is indicative thatthe test compound modulates the activity of the subject polypeptide. Incertain instances, the test compound agonizes the activity of thesubject polypeptide, and in other instances, the test compoundantagonizes the activity of the subject polypeptide.

In other embodiments, a defined transformed cell type in culture may beused to assess 4E activity. Such a cell type may be produced throughartificial or natural over-expression of one or more oncogenes. Forexample, the cell type may overexpress 4E, or a protein that is ancomponent of the 4E regulon that affects cellular transport,transformation and proliferation, such as myc, cyclin D, etc.

Screening of modulators using cell lines possessing over-expressedoncogenes may be accomplished through the analysis of any one orcombinations of the following: (1) 4E expression, protein level,cellular proliferation rate; (2) myc expression, protein level,nuclear/cytoplasmic ratio, cellular proliferation rate; (3) cyclin D1expression, protein level, nuclear/cytoplasmic ratio, cellularproliferation rate; (4) inhibition of cyclin D1 or other reguloncomponent mRNA transport (nucleus to cytoplasm) and/or cyclin D1 orother regulon component mRNA translation; (5) inhibition of cyclin D1 orother regulon component transcription; (6) inhibition of 4E-SE regulatedmRNA transport (nucleus to cytoplasm) and/or 4E-SE mRNA translation; (7)inhibition of 4E-SE regulated mRNA gene transcription.

Once identified, a potential modulator may be used as a model structure,and analogs to the compound can be obtained. The analogs are thenscreened for their ability to modulate 4E activity as described above.

In a related approach, iterative drug design is used to identifymodulators of 4E activity. Iterative drug design is a method foroptimizing associations between a protein and a modulator by determiningand evaluating the three dimensional structures of successive sets ofprotein/modulator complexes. In iterative drug design, crystals of aseries of protein/modulator complexes are obtained and then thethree-dimensional structures of each complex is solved. Such an approachprovides insight into the association between the proteins andmodulators of each complex. For example, this approach may beaccomplished by selecting modulators with inhibitory activity, obtainingcrystals of this new protein/modulator complex, solving the threedimensional structure of the complex, and comparing the associationsbetween the new protein/modulator complex and previously solvedprotein/modulator complexes. By observing how changes in the modulatoraffected the protein/modulator associations, these associations may beoptimized.

Further provided are pharmaceutical compositions comprising theabove-described compounds of Formulas I and II and/or the additionalcompounds described herein. In one aspect, the present inventionprovides pharmaceutically acceptable compositions which comprise atherapeutically-effective amount of one or more of the compoundsdescribed above, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. In another aspect,certain embodiments, the compounds of the invention may be administeredas such or in admixtures with pharmaceutically acceptable carriers andmay also be administered in conjunction with other agents. Conjunctive(combination) therapy thus includes sequential, simultaneous andseparate, or co-administration of the active compound in a way that thetherapeutical effects of the first administered one is not entirelydisappeared when the subsequent is administered.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art. While it is possible for a compoundof the present invention to be administered alone, it is preferable toadminister the compound as a pharmaceutical formulation (composition).The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals. In certain embodiments,a prodrug form of a compound of Formulas I or II comprises thepharmaceutical compositions of the present invention.

As described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for buccal,sublingual, and systemic absorption, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally. In one embodiment, the pharmaceutical compositions areformulated for parenteral administration. In one embodiment, thepharmaceutical composition is formulated for intraarterial injection. Inanother embodiment, the pharmaceutical compositions are formulated forsystemic administration.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants may also be present in the compositions.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which maybe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which may be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect. Incertain embodiments, the carrier material is covalently linked to thecompound or other agent in the composition.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Besides inert diluents, theoral compositions may also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming and preservative agents. Suspensions, in addition to theactive compounds, may contain suspending agents as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate or cyclodextrinand its salts, and/or any of the following: (1) fillers or extenders,such as starches, lactose, sucrose, glucose, mannitol, and/or silicicacid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol, glycerol monostearate, and non-ionic surfactants; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof, and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-shelledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets, and other soliddosage forms of the pharmaceutical compositions of the presentinvention, such as dragees, capsules, pills and granules, may optionallybe scored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.They may also be formulated so as to provide slow or controlled releaseof the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile,cyclodextrin and its salts, other polymer matrices, liposomes and/ormicrospheres. They may be formulated for rapid release, e.g.,freeze-dried. They may be sterilized by, for example, filtration througha bacteria-retaining filter, or by incorporating sterilizing agents inthe form of sterile solid compositions which may be dissolved in sterilewater, or some other sterile injectable medium immediately before use.These compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich may be used include polymeric substances and waxes. The activeingredient may also be in micro-encapsulated form, if appropriate, withone or more of the above-described excipients.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required. The ointments, pastes, creams and gels may contain, inaddition to an active compound of this invention, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof. Powders andsprays can contain, in addition to a compound of this invention,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents. These compositions may also containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. Prevention of the action of microorganisms upon thesubject compounds may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

C. Therapeutic Use of Small Molecule Compositions of Section B

The levels of compounds of Formulas I and II and/or the additionalcompounds described herein required to effect the inhibition of elevated4E activity within cells, tissues, tumors and/or cancers is micromolar.Thus the therapeutic level at which Ribavirin inhibits 4E activity is500-fold less than the quantities previously described that providemechanistically for the use of compounds of Formulas I and II as aco-therapy with interferon for the treatment of hepatitis. Moreover,compounds of Formulas I and II that are active in inhibiting 4E activitywithin cells, tissues, tumors and/or cancers represent a distinctiveshape and charge space from those active at millimolar levels in concertwith the co-administration of IFN for the treatment of hepatitis.Indeed, in mouse models of metastases by Hairisi and colleagues,Ribavirin was observed to reduce liver metastatic disease (Jeney, A., etal. (2006) Magy. Onkol. 50:93-100).

Accordingly, we have discovered that compounds of Formulas I and II willinhibit the growth, proliferation and seeding of new/metastatic cancerswithin individuals, thereby either permitting natural/endogenousimmuno-logical processes within the patient to attack and affect thecomplete removal of the tumor; alternatively cancer therapy based on thecompounds of Formulas I and II will permit the long-term management of4E activity cancers through chronic administration of compounds ofFormulas I and II at doses 500-fold less than those required for thetreatment of hepatitis and far below levels that may be toxic during thechronic administration of compounds of Formulas I and II required toaffect the long-term management of disease conditions where elevated 4Eactivities are operative, including cancers, tumors and the like.

Prevention of the continued growth and proliferation of existing cancers(rendering them static) represents a huge advance in the management ofthe many cancers exhibiting elevated 4E activity—including head/neck,breast prostate, lung, cervix, among others.

In addition, as many of the angiogenic and autocrine factors produced bycancers are 4E regulated at the translational level, therapy comprisingthe administration of compounds of Formulas I and II therapy isanticipated to inhibit the process of angiogenesis required for thecontinued growth and establishment of metastatic cancers.

Thus, the administration of compounds of Formulas I and II may providetherapeutic interventions that are determined to be appropriate foradditional non-cancerous conditions where elevated 4E activity isdetermined to be a causative factor.

Accordingly, provided are methods of treating subjects in need thereofwith compositions comprising compounds of Formulas I and II and/or theadditional compounds described herein. The compounds of Formulas I andII inhibit elevated 4E activity, e.g. by targeting 4E. In particular,they inhibit cellular proliferation and 4E regulon activity in cells,tissues and mammals, e.g., where there exists a condition of elevated 4Eactivity. Thus, the compounds of Formulas I and II are expected to actas inhibitors of cancer cell proliferation, especially in cells tissues,mammals where there exists a condition of elevated 4E activity. Thus,they may be used in methods of inhibiting elevated 4E activity in acell, tissue or mammal and in methods of inhibiting cellularproliferation in a cell, tissue or mammal.

Compositions comprising compounds of Formulas I and II and/or theadditional compounds described herein also may be used in treating asubject having cancer. The compositions may be especially useful intreating conditions caused by elevated 4E activity, for example, cancer,as defined above. The compositions comprising compounds of Formulas Iand II and/or the additional compounds described herein may also be usedas inhibitors and/or dose-dependent regulators of any of the cancertherapeutic vectors disclosed below, as will be described in more detailbelow.

In certain embodiments, the compositions comprising compounds ofFormulas I and II may inhibit the metastatic phenotype represented byelevated 4E activity, thus resulting in prophylactic anti-metastasis.

In certain embodiments, therapies comprising the use of compounds ofFormulas I and II may also comprise the use of other cancertherapeutics, such as interferon, kinase inhibitors, gene therapyvectors as described further below in Section D and/or chemotherapeuticagents, biologics and cytotoxins described immediately below.

The term “chemotherapeutic agent” refers to any small molecule orcomposition used to treat disease caused by a foreign cell or malignantcell, such as a tumor cell. Non-limiting examples of chemotherapeuticagents include agents that disrupt DNA synthesis, are inhibitors oftopoisomerase I, are alkylating agents, or are plant alkaloids. The term“agent that disrupts DNA synthesis” refers to any molecule or compoundable to reduce or inhibit the process of DNA synthesis. Examples ofagents that disrupt DNA synthesis include but are not limited tonucleoside analogs such as pyrimidine or purine analogs, including, forexample but not limited to, gemcitabine or alternatively anthracyclinecompounds, including for example but not limited to, adriamycin,daunombicin, doxorabicin, and idambicin and epipodophyllotoxins such asetoposide and teniposide. The term “topoisomerase I inhibitor” refers toa molecule or compound that inhibits or reduces the biological activityof a topoisomerase I enzyme. Including for example, but not limited to,camptosar. The term “alkylating agent” refers to any molecule orcompound able to react with the nucleophilic groups of (for examples,amines, alcohols, phenols, organic and inorganic acids) and thus addalkyl groups (for example, ethyl or methyl groups) to another moleculesuch as a protein or nucleic acid. Examples of alkylating agents used aschemotherapeutic agents include bisulfan, chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine, melphalan, thiotepa,various nitrosourea compounds, and platinum compounds such as cisplatinand carboplatin. The term “plant alkaloid” refers a compound belongingto a family of alkaline, nitrogen-containing molecules derived fromplants that are biologically active and cytotoxic. Examples of plantalkoids include, but are not limited to, taxanes such as taxol,docetaxel and paclitaxel and vincas such as vinblastine, vincristine,and vinorelbine.

Biologics may include antibodies or antigen binding fragments thereof,that bind to a targetable component of a tumor cell, tumor vasculatureor tumor stroma. A “targetable component” of a tumor cell, tumorvasculature or tumor stroma, is preferably a surface-expressed,surface-accessible or surface-localized component, although componentsreleased from necrotic or otherwise damaged tumor cells or vascularendothelial cells may also be targeted, including cytosolic and/ornuclear tumor cell antigens.

Biologics may also include anti-tumor cell immunotoxins or coaguligandssuch as antibodies exemplified by the group consisting of B3 (ATCC HB10573), 260F9 (ATCC HB 8488), D612 (ATCC HB 9796) and KS1/4, said KS1/4antibody obtained from a cell comprising the vector pGKC2310 (NRRLB-18356) or the vector pG2A52 (NRRL B-18357). Biologics may beanti-tumor stroma immunotoxins or coaguligands, for example, antibodiesthat bind to a connective tissue component, a basement membranecomponent or an activated platelet component; as exemplified by bindingto fibrin, RIBS or LIBS.

Biologics such as anti-tumor vasculature immunotoxins or coaguligandsmay also comprise ligands, antibodies, or fragments thereof, that bindto a surface-expressed, surface-accessible or surface-localizedcomponent of the blood transporting vessels, preferably the intratumoralblood vessels, of a vascularized tumor. Such antibodies include thosethat bind to surface-expressed components of intratumoral blood vesselsof a vascularized tumor, including aminophospholipids themselves, andintratumoral vasculature cell surface receptors, such as endoglin (TEC-4and TEC-11 antibodies), a TGF.beta. receptor, E-selectin, P-selectin,VCAM-1, ICAM-1, PSMA, a VEGF/VPF receptor, an FGF receptor, a TIE,.alpha.sub.v.beta.sub.3 integrin, pleiotropin, endosialin and MHC ClassII proteins. The antibodies may also bind to cytokine-inducible orcoagulant-inducible components of intratumoral blood vessels.

Other anti-tumor vasculature immunotoxins or coaguligands may compriseantibodies, or fragments thereof, that bind to a ligand or growth factorthat binds to an intratumoral vasculature cell surface receptor. Suchantibodies include those that bind to VEGF/VPF (GV39 and GV97antibodies), FGF, TGF.beta., a ligand that binds to a TIE, atumor-associated fibronectin isoform, scatter factor/hepatocyte growthfactor (HGF), platelet factor 4 (PF4), PDGF and TIMP. The antibodies, orfragments thereof, may also bind to a ligand:receptor complex or agrowth factor:receptor complex, but not to the ligand or growth factor,or to the receptor, when the ligand or growth factor or the receptor isnot in the ligand:receptor or growth factor:receptor complex.

Cytotoxic agents such as plant-, fungus- or bacteria-derived toxins(immunotoxins). Ricin A chain, deglycosylated ricin A chain, gelonin andangiopoietins may also be used in combination therapies.

Dosage may be based on the amount of the composition per kg body weightof the patient. Other amounts will be known to those of skill in the artand readily determined. Alternatively, the dosage of the subjectinvention may be determined by reference to the plasma concentrations ofthe composition. For example, the maximum plasma concentration (Cmax)and the area under the plasma concentration-time curve from time 0 toinfinity (AUC (0-4)) may be used. Dosages for the present inventioninclude those that produce the above values for C_(max) and AUC (0-4)and other dosages resulting in larger or smaller values for thoseparameters.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above.

Dosage levels of between about 0.01 and about 5 mg/kg body weight perday, preferably between about 0.1 and about 2.5 mg/kg body weight perday of the modulators described herein are useful for the prevention andtreatment of disease and conditions related to 4E activity. The amountof active ingredient that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration.

The precise time of administration and amount of any particular compoundthat will yield the most effective treatment in a given patient willdepend upon the activity, pharmacokinetics, and bioavailability of aparticular compound, physiological condition of the patient (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage and type of medication), route ofadministration, and the like. The guidelines presented herein may beused to optimize the treatment, e.g., determining the optimum timeand/or amount of administration, which will require no more than routineexperimentation consisting of monitoring the subject and adjusting thedosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. Treatment, includingsupplement, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters, the first such reevaluation typicallyoccurring at the end of four weeks from the onset of therapy, andsubsequent reevaluations occurring every four to eight weeks duringtherapy and then every three months thereafter. Therapy may continue forseveral months or even years, with a minimum of one month being atypical length of therapy for humans. Adjustments to the amount(s) ofagent administered and possibly to the time of administration may bemade based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The combined use of several compounds of the present invention, oralternatively other chemotherapeutic agents, may reduce the requireddosage for any individual component because the onset and duration ofeffect of the different components may be complimentary. In suchcombined therapy, the different active agents may be delivered togetheror separately, and simultaneously or at different times within the day.Toxicity and therapeutic efficacy of subject compounds may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 and the ED50. Compositions thatexhibit large therapeutic indices are preferred. Although compounds thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets the compounds to the desired site in orderto reduce side effects.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany supplement, or alternatively of any components therein, liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For agents of the present invention, the therapeuticallyeffective dose may be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information may be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

D. Compositions for Gene Therapy

Provided also are compositions comprising gene therapeutic vectors andviruses that, among other things, enhance regulation of mRNA nuclear tocytoplasmic transport and/or mRNA translation. The vectors and virusesmay comprise nucleic acids, e.g., DNA constructs or mRNAs, encodingproteins contained within gene therapeutic vector/virus required forvector and/or viral replication and/or lysis, nucleic acids, e.g.,mRNAs, encoding therapeutic proteins required for gene therapeuticactivity including but not limited to toxins, lytic peptides and/orproteins and/or processes and therapeutic proteins including but notlimited to prodrug converting enzymes (aka suicide genes),anti-angiogenic proteins, apoptosis cascade enzymes, tumor suppressors,cytokines and immunologically active proteins, RNAi anti-sense, and thelike.

We explored the possibility that in the nucleus that 4E recognition ofmRNA is fundamentally different than in the cytoplasm and identified a100-nt sequence from the cyclin D1 3-UTR which sensitizes this mRNA to4E in the nucleus, is required for nuclear to cytoplasmic transport ofmRNAs containing this sequence and which participates in 4E mediatedcellular transformation.

Provided accordingly is the 4E-SE sequence in FIG. 14 c (SEQ ID NO:1).The sequence may serve as a 3′UTR, 5′UTR, or other control element of anmRNA or DNA construct, which may in some embodiments serve to make thenuclear to cytoplasmic transport and/or translation of said mRNAdependent upon the presence of elevated 4E activity. Allowing for thedegeneracy of the genetic code, sequences that have at least about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to SEQ ID NO: 1are polynucleotides that may be used in the constructs and vectorsdescribed herein, provided that they include the minimal ˜50 nucleotideregion of FIG. 19 a.

Such constructs and vectors comprising 4E-SE may have the followingproperties and uses. In certain embodiments, vector and viralreplication may be placed under 4E-SE regulation. In other embodiments,vector/viral induced cellular lysis may be placed under 4E-SE controlelement regulation. In still other embodiments, nuclear to cytoplasmictransport and/or translation of one or more vector/virally encoded mRNAsmay be placed under 4E-SE control element regulation. In otherembodiments, the nuclear to cytoplasmic transport and/or translation ofone or more of vector/virally encoded mRNAs required for cellular lysismay be placed under 4E-SE regulation.

Any of above described vectors may contain an intron encoding an RNA ormRNA as appropriate for one or more of the following: toxins; lyticpeptides and/or proteins/processes; regulators of angiogenesis,apoptosis cascade enzymes, tumor suppressors, cytokines andimmunologically active proteins (including but not limited tointerferon, GMCSF, GCSF and others which have been demonstrated toenhance host immune response to tumors yet where systemic delivery isoften associated with significant side/detrimental clinical effects),RNAi, RNA anti-sense, and the like.

The resultant vectors may provide for enhanced selectivity andrestriction of gene therapeutic expression, enhanced selectivity andrestriction of gene therapeutic expression to environments possessingelevated 4E activity, and/or enhanced selectivity and restriction ofgene therapeutic expression to environments possessing elevated 4Eactivity thereby rendering the transport and/or translation subject toinhibition by any of the compounds of Formulas I and II disclosed above.

Inhibition of the gene therapeutic activity of any of the above vectorsvia the administration of any of the compounds of Formulas I and IIprovides a method of inhibiting gene therapeutic activity and therebyhalting the therapeutic process should the administration of the genetherapeutic provide an unfavorable impact upon the subject mammal towhich it has been administered. Targeted presentation within elevated 4Ecellular and/or tissue environments provides enhanced selectivity andspecificity to cells and/or tissues possessing an elevated 4Eenvironment. Improved targeting of such gene therapeutics to increasethe efficacy of such agents and/or to reduce systemic toxicity oftenreported subsequent to the systemic (non-targeted) administration ofthese classes of agents. Thus, the compounds of Formulas I and II mayserve as inhibitors and/or dose-dependent regulators of any of thevectors disclosed herein. Various embodiments of combination therapiescomprising vectors and the compounds of Formulas I and II are describedin the next section.

Gene therapeutic vectors exhibiting enhanced efficacy profiles may alsocomprise any of the vectors described above containing an intronencoding an mRNA encoding an improved prodrug metabolizing/convertingenzyme. The doubly improved prodrug metabolizing/converting enzyme maybe selected from among (but not limited by) those disclosed by Black,Loeb and co-workers (thymidine kinases and/or cytosine deaminase) whichprovide but are not limited to possessing enhanced affinity and/orselectivity for prodrug substrate, and/or rate of prodrug conversion toactive desired cytotoxic product and the like.

Any gene therapeutic intron or viral lytic replication element asdisclosed herein may be replaced with any of the following therapeuticintrons or RNAs including but without limitation to the followingclasses of agents: (1) anti-sense RNA, RNAi, ribozymes, single chainantibodies targeted and designed to inhibit either the RNAs and mRNAsexpressed within elevated 4E environments and/or inhibit the activity ofthe translation products of mRNAs expressed within elevated 4Eenvironments in cells, tissues and animals and (2) RNAi, antisenseand/or ribozymes targeted to 4E-SE control element sequences. Such genetherapeutics are referred to as “inhibitory gene therapeutics” herein.They may be administered in vitro or in vivo using methods known in theart.

Constructs and vectors may contain (a) a transcriptional control elementas described above, and (b) flanking DNA sequence from a target genepermitting the homologous recombination of the transcriptional controlelement into a host cell in association with the target gene. In otherembodiments the construct or vector contains a desired gene and flankingDNA sequence from a target locus permitting the homologous recombinationof the target gene into the desired locus. The construct or vector mayalso contain the responsive transcriptional control element, or theresponsive element may be provided by the locus.

The constructs or vectors may also contain a selectable markerpermitting transfection of the constructs into host cells and selectionof transfectants containing the construct or vector. This inventionfurther encompasses DNA vectors containing such constructs, whether forepisomal transfection or for integration into the host cell chromosomes.The vector may be a viral vector, including for example an adeno-, adenoassociated- or retroviral vector.

Vectors, such as viral vectors have been used in the prior art tointroduce genes into a wide variety of different target cells. Typicallythe vectors are exposed to the target cells so that transformation cantake place in a sufficient proportion of the cells to provide a usefultherapeutic or prophylactic effect from the expression of the desiredpolypeptide. The transfected nucleic acid may be permanentlyincorporated into the genome of each of the targeted cells, providinglong lasting effect, or alternatively the treatment may have to berepeated periodically.

A variety of vectors, both viral vectors and plasmid vectors are knownin the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular,a number of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpes viruses includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have employed disabled murine retroviruses.

Several recently issued patents are directed to methods and compositionsfor performing gene therapy. See U.S. Pat. Nos. 6,168,916; 6,135,976;5,965,541 and 6,129,705. Each of the foregoing patents is incorporatedby reference herein.

E. Additional Combination Therapies and Co-Therapies

In addition to the combination therapies described in Section D, thecompounds of Formulas I and II may serve as inhibitors and/ordose-dependent regulators of any of the inhibitory gene therapeuticsdisclosed above. For example, the compounds of Formulas I and II mayserve as inhibitors (whether present alone or in concert) of 4E-SE asdescribed above, including when 4E-SE is a control element of an mRNA.

Further, in other embodiments, the compounds of Formulas I and II mayserve as inhibitors (whether present alone or in concert) of mRNAtransport and/or translation. For example, the compounds of Formulas Iand II may serve as inhibitors (whether present alone or in concert) ofthe transport and/or translation of mRNAs containing 4E-SE as describedabove, including when 4E-SE is a control element of an mRNA.

In still other embodiments, the compounds of Formulas I and II may serveas inhibitors of vector or viral replication under 4E-SE regulation.Further, in other embodiments, the compounds of Formulas I and II mayserve as inhibitors of vector or viral induced cellular lysis under4E-SE regulation. In yet another embodiment, the compounds of Formulas Iand II may serve as inhibitors of the nuclear to cytoplasmic transportand/or translation of one or more vector or virally encoded mRNAs, e.g.,such as those required for cellular lysis, under 4E-SE regulation and/orof one or more therapeutic gene mRNAs or introns contained within any ofthe above vectors under 4E-SE regulation.

The gene therapeutics described herein and small molecule inhibitors ofmRNA nuclear to cytoplasmic transport and/or protein translationalprocesses (such as compounds of Formulas I and II) may be used togetheror in concert to inhibit elevated 4E activity within cells, tissues andmammals, to inhibit cellular proliferation within cells, tissues andmammals (e.g., those possessing elevated 4E activity), and/or to inhibitcellular proliferation within cancers and/or tumors (e.g., thosepossessing elevated 4E activity).

Such enhanced methods and compositions for the treatment of cellproliferative disorders in which there exists elevated 4E activity whereadministration of small molecules and/or gene therapeutics alone or inconcert may fail to eradicate the cell proliferative disorder or canceror tumor, yet inhibit its continued proliferation and expansion, thusproviding either an entre for the host immune system to eradicate thecell proliferative disorder or tumor or cancer; or serving to make thecell proliferative disorder or tumor or cancer manageable through theroutine administration of small molecules with or without the periodicco-administration of additional systemic agents/biologics, and/or theperiodic co-administration of any one/more of the gene therapeuticsmethodologies disclosed herein. Where conditions are arrived at in theabove embodiment so to render the cell proliferative disorder, cancer ortumor “phenotypically revertant” thereby transitioning the diseaseprocess from life threatening to a chronic disease state.

F. Protein Expression

Any of the gene therapeutics and/or gene diagnostics disclosed hereinmay be used to provide to the production of therapeutic proteins withineukaryotic protein expression systems such the following which areprovided only as a non-exclusive list of the various expression systems(mammalian cells, insect cells and/or yeast and the like) where theselective nuclear to cytoplasmic transport and translation of proteinsunder 4E-SE control (as disclosed herein) within a cellular environmentin which there exists elevated 4E activity.

The compounds of Formulas I and II may serve as inhibitors and/ordose-dependent regulators of any of the gene therapeutics disclosedherein. In vitro therapeutic protein production systems in which mRNAswhich are translated into structural proteins which are detrimental tothe isolation of therapeutic protein(s) of interest which can beimproved via the introduction of 4E-SE control elements within thesedetrimental elements such that prior to the commencement of therapeuticprotein isolation the addition of compounds of Formulas I and II acts toinhibit the transport and translation of the detrimental mRNAs. Theinhibition (or the removal from compounds of Formulas I and II-mediatedinhibition) of non-therapeutic protein synthesis via the addition (orremoval) of compounds of Formulas I and II serves to enhance thesubsequent isolation of the therapeutic protein of interest.

Protein expression systems in which the therapeutic protein of interestis under the control of an inducible promoter and where compounds ofFormulas I and II can either serve to enhance the basal repressionmediated by the inducible promoter (via inhibition of mRNA transport andtranslation) may serve to regulate in a dose-dependent fashion mRNAtransport and translation post additional on the inducible promoter‘ligand’ or inhibit transport and translation of the therapeutic proteinalso post-induction.

Within certain embodiments, expression vectors are employed. Expressionrequires that appropriate signals be provided in the vectors, whichinclude various regulatory elements, such as enhancers/promoters fromboth viral and mammalian sources that drive expression of the genes ofinterest in host cells. Elements designed to optimize messenger RNAstability and translatability in host cells are also defined. Theconditions for the use of a number of dominant drug selection markersfor establishing permanent, stable cell clones expressing the productsare also provided, as is an element that links expression of the drugselection markers to expression of the polypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a polynucleotide codingfor a gene product in which part or all of the polynucleotide encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the polynucleotide encoding a gene of interest.

The polynucleotide encoding a gene product may be under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase “under transcriptional control” means that the promoter is inthe correct location and orientation in relation to the polynucleotideto control RNA polymerase initiation and expression of the gene.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30 to 110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of apolynucleotide sequence of interest is not believed to be important, solong as it is capable of directing the expression of the polynucleotidein the targeted cell. Thus, where a human cell is targeted, it ispreferable to position the polynucleotide coding region adjacent to andunder the control of a promoter that is capable of being expressed in ahuman cell. Generally speaking, such a promoter might include either ahuman or viral promoter. The use of viral or mammalian cellular orbacterial phage promoters which are well-known in the art to achieveexpression of a coding sequence of interest is contemplated as well,provided that the levels of expression are sufficient for a givenpurpose. By employing a promoter with well-known properties, the leveland pattern of expression of the protein of interest followingtransfection or transformation can be optimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcriptionin specific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate.

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. Cytokine and inflammatory protein responsive promoters thatcould be used include K and T Kininogen, c-fos, TNF-alpha, C-reactiveprotein, haptoglobin, serum amyloid A2, C/EBP alpha, IL-1, IL-6,Complement C3, IL-8, alpha-1 acid glycoprotein, alpha-1 antitypsin,lipoprotein lipase, angiotensinogen, fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin.

Tumor specific promoters such as osteocalcin; hypoxia-responsive element(HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may alsobe used to regulate gene expression in tumor cells. Other promoters thatcould be used according to the present invention includeLac-regulatable, chemotherapy inducible (e.g. MDR), and heat(hyperthermia) inducible promoters, radiation-inducible (e.g., EGR),Alpha-inhibin, RNA pol III tRNA met and other amino acid promoters, U1snRNA, MC-1, PGK, .beta.-actin and .alpha.-globin. Many other promotersthat may be useful are listed in Walther and Stein (1996) J. Mol. Med,74:379-392.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are frequentlyoverlapping and contiguous, often seeming to have a very similar modularorganization.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

In certain embodiments of the invention, the cells containpolynucleotide constructs of the present invention, and a cell may beidentified in vitro or in vivo by including a marker in the expressionconstruct. Such markers will confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. Usually the inclusion of a drug selection marker aids incloning and in the selection of transformants, for example, genes thatencode neomycin, puromycin, hygromycin, DHFR, GPT, HPRT, zeocin, andhistidinol are useful selectable markers. Alternatively, enzymes such asherpes simplex virus thymidine kinase (tk) or chloramphenicolacetyltransferase (CAT) may be employed. Immunologic markers also can beemployed. The selectable marker employed is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the polynucleotide encoding a gene product. Further examples ofselectable markers are well known to one of skill in the art.

In certain embodiments of the invention, internal ribosome binding sites(IRES) elements are used to create multigene, or polycistronic,messages. IRES elements are able to bypass the ribosome scanning modelof 5′ methylated Cap dependent translation and begin translation atinternal sites (Pelletier and Sonenberg, (1988) Nature 334:320 325).IRES elements from two members of the picanovirus family (polio andencephalomyocarditis) have been described, as well an IRES from amammalian message. IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame is accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins, andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

There are a number of ways to introduce expression vectors into cells.In certain embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis, to integrate into host cell genome and express viral genesstably and efficiently have made them attractive candidates for thetransfer of foreign genes into mammalian cells. The first viruses usedas gene vectors were DNA viruses including the papovaviruses (simianvirus 40, bovine papilloma virus, and polyoma) and adenoviruses. Thesehave a relatively low capacity for foreign DNA sequences and have arestricted host spectrum. Furthermore, their oncogenic potential andcytopathic effects in permissive cells raise safety concerns. They canaccommodate only up to 8 kb of foreign genetic material but can bereadily introduced in a variety of cell lines and laboratory animals.

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription. The resultingDNA then stably integrates into cellular chromosomes as a provirus anddirects synthesis of viral proteins. The integration results in theretention of the viral gene sequences in the recipient cell and itsdescendants. The retroviral genome contains three genes, gag, pol, andenv that code for capsid proteins, polymerase enzyme, and envelopecomponents, respectively. A sequence found upstream from the gag genecontains a signal for packaging of the genome into virions. Two longterminal repeat (LTR) sequences are present at the 5′ and 3′ ends of theviral genome. These contain strong promoter and enhancer sequences andare also required for integration in the host cell genome.

In order to construct a retroviral vector, a polynucleotide encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed. When a recombinant plasmidcontaining a cDNA, together with the retroviral LTR and packagingsequences is introduced into this cell line (by calcium phosphateprecipitation for example), the packaging sequence allows the RNAtranscript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media. The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells.

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin. Usingantibodies against major histocompatibility complex class I and class IIantigens, they demonstrated the infection of a variety of human cellsthat bore those surface antigens with an ecotropic virus in vitro.

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes. Another concern with the use ofdefective retrovirus vectors is the potential appearance of wild-typereplication-competent virus in the packaging cells. This can result fromrecombination events in which the intact-sequence from the recombinantvirus inserts upstream from the gag, pol, env sequence integrated in thehost cell genome. However, new packaging cell lines are now availablethat should greatly decrease the likelihood of recombination.

Lentiviruses can also be used as vectors in the present application. Inaddition to the long-term expression of the tran-transgene provided byall retroviral vectors, lentiviruses present the opportunity totransduce nondividing cells and potentially achieve regulatedexpression. The development of lentiviral vectors requires the design oftransfer vectors to ferry the transgene with efficient encapsidation ofthe transgene RNA and with full expression capability, and of apackaging vector to provide packaging machinery in trans but withouthelper virus production. For both vectors, a knowledge of packagingsignal is required-the signal to be included in the transfer vector butexcluded from the packaging vector. Exemplary human lentiviruses arehuman immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). HIV-2is likely better suited for gene transfer than HIV-1 as it is lesspathogenic and thus safer during design and production; its desirablenuclear import and undesirable cell-cycle arrest functions aresegregated on two separate genes.

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb. In contrastto retrovirus, the adenoviral infection of host cells does not result inchromosomal integration because adenoviral DNA can replicate in anepisomal manner without potential genotoxicity. Also, adenoviruses arestructurally stable, and no genome rearrangement has been detected afterextensive amplification. Adenovirus can infect virtually all epithelialcells regardless of their cell cycle stage. So far, adenoviral infectionappears to be linked only to mild disease such as acute respiratorydisease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100 200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off. Theproducts of the late genes, including the majority of the viral capsidproteins, are expressed only after significant processing of a singleprimary transcript issued by the major late promoter (MLP). The MLP,(located at 16.8 m.u.) is particularly efficient during the late phaseof infection, and all the mRNAs issued from this promoter possess a5′-tripartite leader (TPL) sequence which makes them preferred mRNAs fortranslation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins. Since the E3 regionis dispensable from the adenovirus genome, the current adenovirusvectors, with the help of 293 cells, carry foreign DNA in either the E1,the D3 or both regions. In nature, adenovirus can package approximately105% of the wild-type genome, providing capacity for about 2 extra kb ofDNA. Combined with the approximately 5.5 kb of DNA that is replaceablein the E1 and E3 regions, the maximum capacity of the current adenovirusvector is under 7.5 kb, or about 15% of the total length of the vector.More than 80% of the adenovirus viral genome remains in the vectorbackbone and is the source of vector-borne cytotoxicity. Also, thereplication deficiency of the E1-deleted virus is incomplete. Forexample, leakage of viral gene expression has been observed with thecurrently available vectors at high multiplicities of infection (MOI).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Methods for culturing 293 cells and propagating adenovirus may includegrowing natural cell aggregates by inoculating individual cells into 1liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100200 ml of medium. Following stirring at 40 rpm, the cell viability isestimated with trypan blue. In another format, Fibra-Cel microcarriers(Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cellinoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml)in a 250 ml Erlenmeyer flask and left stationary, with occasionalagitation, for 1 to 4 hours. The medium is then replaced with 50 ml offresh medium and shaking initiated. For virus production, cells areallowed to grow to about 80% confluence, after which time the medium isreplaced (to 25% of the final volume) and adenovirus added at an MOI of0.05. Cultures are left stationary overnight, following which the volumeis increased to 100% and shaking commenced for another 72 hours.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10.sup.9 10.sup.11 plaque-forming units per ml, and theyare highly infective. The life cycle of adenovirus does not requireintegration into the host cell genome. The foreign genes delivered byadenovirus vectors are episomal and, therefore, have low genotoxicity tohost cells. No side effects have been reported in studies of vaccinationwith wild-type adenovirus, demonstrating their safety and therapeuticpotential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression andvaccine development. Recently, animal studies suggested that recombinantadenovirus could be used for gene therapy (Stratford-Perricaudet andPerricaudet, (1991) In: Human Gene Transfer, Eds, O. Cohen-Haguenauerand M. Boiron, Editions John Libbey Eurotext, France, pp. 51 61;Stratford-Perricaudet et al. (1990) Hum. Gene Ther., 1:241 256; and Richet al. (1993) Hum. Gene Ther., 4:461 476). Studies in administeringrecombinant adenovirus to different tissues include tracheainstillation, muscle injection, peripheral intravenous injections andstereotactic inoculation into the brain.

Adeno-associated virus (AAV) utilizes a linear, single-stranded DNA ofabout 4700 base pairs. Inverted terminal repeats flank the genome. Twogenes are present within the genome, giving rise to a number of distinctgene products. The first, the cap gene, produces three different virionproteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene,encodes four non-structural proteins (NS). One or more of these rep geneproducts is responsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, inmap-units, in the genome. These are, from left to right, p5, p19 andp40. Transcription gives rise to six transcripts, two initiated at eachof three promoters, with one of each pair being spliced. The splicesite, derived from map units 42 46, is the same for each transcript. Thefour non-structural proteins apparently are derived from the longer ofthe transcripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of an AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as psub201, whichcontains a modified AAV genome, or by other methods known to the skilledartisan, including but not limited to chemical or enzymatic synthesis ofthe terminal repeats based upon the published sequence of AAV. Theordinarily skilled artisan can determine, by well-known methods such asdeletion analysis, the minimum sequence or part of the AAV ITRs which isrequired to allow function, i.e. stable and site-specific integration.The ordinarily skilled artisan also can determine which minormodifications of the sequence can be tolerated while maintaining theability of the terminal repeats to direct stable, site-specificintegration.

AAV-based vectors have proven to be safe and effective vehicle for genedelivery in vitro, and these vectors are now being developed and testedin pre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo.

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Baichwal and Sugden (1986) In: Gene Transfer, Kucherlapati R, ed., NewYork, Plenum Press, 117 148) adeno-associated virus (AAV) (Baichwal andSugden, 1986) moloney murine leukemia virus (MoMuLV); VSV-G typeretroviruses (U.S. Pat. No. 5,817,491, specifically incorporated hereinby reference), papovaviruses such as JC, SV40, polyoma (U.S. Pat. No.5,624,820, specifically incorporated herein by reference) Epstein-BarrVirus (EBV); papilloma viruses (U.S. Pat. No. 5,674,703, specificallyincorporated herein by reference), and more particularly, bovinepapilloma virus type I (BPV; U.S. Pat. No. 4,419,446, incorporatedherein by reference); poliovirus herpesviruses and other human andanimal viruses may be employed. These viruses offer several attractivefeatures for various mammalian cells.

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsulated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation, DEAE-dextran,electroporation, direct microinjection, DNA-loaded liposomes andlipofectamine-DNA complexes, cell sonication, gene bombardment usinghigh velocity microprojectiles, and receptor-mediated transfection. Someof these techniques may be successfully adapted for in vivo or ex vivouse.

Once the expression construct has been delivered into the cell thepolynucleotide encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the polynucleotideencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the polynucleotide may be stably maintained in the cell asa separate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the polynucleotide remains is dependent on the type ofexpression construct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. It isenvisioned that DNA encoding a gene of interest may also be transferredin a similar manner in vivo and express the gene product.

In still another embodiment of the invention, transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them. Several devices for accelerating smallparticles have been developed. One such device relies on a high voltagedischarge to generate an electrical current, which in turn provides themotive force. The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo. This may require surgical exposure ofthe tissue or cells, to eliminate any intervening tissue between the gunand the target organ, i.e., ex vivo treatment. Again, DNA encoding aparticular gene may be delivered via this method and still beincorporated by the present invention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers. Alsocontemplated are lipofectamine-DNA complexes.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA. In other embodiments, the liposome may becomplexed or employed in conjunction with nuclear non-histonechromosomal proteins (HMG-1). In yet further embodiments, the liposomemay be complexed or employed in conjunction with both HVJ and HMG-1.Since these expression constructs have been successfully employed intransfer and expression of polynucleotides in vitro and in vivo, thenthey are applicable for the present invention. Where a bacterialpromoter is employed in the DNA construct, it also will be desirable toinclude within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver apolynucleotide encoding a particular gene into cells arereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific.

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) andtransferrin. Recently, a synthetic neoglycoprotein, which recognizes thesame receptor as ASOR, has been used as a gene delivery vehicle andepidermal growth factor (EGF) has also been used to deliver genes tosquamous carcinoma cells.

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, epidermal growth factor (EGF) may be used as thereceptor for mediated delivery of a polynucleotide encoding a gene inmany tumor cells that exhibit upregulation of EGF receptor. Mannose canbe used to target the mannose receptor on liver cells. Also, antibodiesto CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma)can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a polynucleotide into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

G. Methods of Diagnosing Elevated 4E Activity

The above-described methods and compositions may be incorporated intodiagnostic methods to detect 4E activity, for example, detect elevated4E levels, expression of 4E, activity or expression of 4E reguloncomponents, etc. Such methods may provide enhanced detection of elevated4E conditions in an animal, tissue or cell, new methods of diagnosing,detecting during surgery, following clinical course of therapeuticefficacy and disease progression/regression and methods for identifyingcompounds and/or biologics that inhibit the transport and or translationany/all/one of gene diagnostics as described herein. For example, amethod for identifying a candidate therapeutic for treating cancer maycomprise: (a) contacting a cell with said candidate therapeutic, (b)determining in the cell pre- and post-contact with said candidatetherapeutic the level of 4E activity or 4E regulon component activity,wherein modulation of the level of 4E activity or 4E regulon componentactivity indicates that the candidate therapeutic may be a therapeuticagent for treating or preventing cancer. This approach may be used tofurther define and/or refine the appropriate human dosing levels insituations where pre- and post-contact samples consist of patientbiopsies, samples, and the like. The candidate therapeutic may be partof a library of candidate therapeutics, for example, one generated usingcombinatorial synthetic methods.

Replacement of any gene therapeutic intron or viral lytic replicationelement as disclosed herein with a gene diagnostic permits theidentification of situations were elevated 4E environments exist withincells and/or tissues. Such gene diagnostic introns can be administeredin vitro or in vivo. The gene diagnostic introns can include withoutlimitation those encoding mRNAs for any of the following: (1) in vivodiagnostic gene administration and methods of imaging as disclosedherein (see above), (2) prodrug metabolizing enzymes that convertappropriate imaging probes (PET probes and the like) thereby serving toconcentrate and localize the imaging agent to the interior of cellsand/or tissues where an elevated 4E environment exists, (3) prodrugmetabolizing enzymes that convert prodrugs (GCV, ACV, 5FC and the like)to active cytotoxic metabolites which are concentrated and localizedwithin environments possessing elevated 4E activity, thereby inhibitingcellular proliferation and/or inducing cell death, (4) fluorescentproteins (green fluorescent protein and the like for example) that serveto identify cells and/or tissues where an elevated 4E environmentexists, (5) beta-galactosidase which when incubated together withappropriate substrates serves to identify cells and/or tissues where anelevated 4E environment exists and (6) viral replication elementsthereby permitting detection of elevated 4E activity through cellularlysis.

Methods for determining the activity of 4E are well known in the art. 4Eactivity as defined herein also includes evaluating 4E regulonactivation, expression and/or activity of components under control ofthe 4E regulon, elevated transport of selected messages (especiallycyclin D1) from the nucleus to the cytoplasm and phosphorylation stateof 4E and levels of e1F4EBP1.

For example, the expression level of 4E regulon components can bedetermined by reverse transcription-polymerase chain reaction (RT-PCR);dot blot analysis; Northern blot analysis, total mRNA by real time PCRand in situ hybridization. Alternatively, the level of 4E reguloncomponents can be analyzed using an appropriate antibody. In certainembodiments, the amount of a 4E regulon component is determined usingantibodies against the 4E regulon component.

In certain embodiments, the level of a protein of interest is determinedby determining its AQUA™ score, e.g., by using the AQUA automatedpathology system. AQUA™ (for Automated Quantitative Analysis) is amethod of analysis of absolute measurement of protein expression insitu. This method allows measurements of protein expression withinsub-cellular compartments that results in a number directly proportionalto the number of molecules expressed per unit area. For example, tomeasure nuclear estrogen receptor (ER), the tissue is “masked” usingkeratin in one channel to normalize the area of tumor and to remove thestromal and other non-tumor material from analysis. Then an image istaken using DAPI to define a nuclear compartment. The pixels within themask and within the DAPI-defined compartment are defined as nuclear. Theintensity of expression of ER is then measured using a third channel.The intensity of that subset of pixels divided by the number of pixels(to normalize the area from spot to spot) to give an AQUA™ score. Thisscore is directly proportional to the number of molecules of ER per unitarea of tumor, as assessed by a standard curve of cell lines with knownlevels of ER protein expression. This method, including details ofout-of-focus light subtraction imaging methods, is described in detailin a Nature Medicine paper (Camp, R. L., Chung, G. G. & Rimm, D. L.Automated subcellular localization and quantification of proteinexpression in tissue microarrays. Nat Med 8, 1323-7 (2002)), as well asU.S. Ser. No. 10/062,308, filed Feb. 1, 2002, both of which referenceare incorporated herein by their entireties.

In other embodiments, methods of detecting the level of expression of 4Eregulon components or other molecule of interest may comprise the use ofa microarray. Arrays are often divided into microarrays and macroarrays,where microarrays have a much higher density of individual probe speciesper area. Microarrays may have as many as 1000 or more different probesin a 1 cm² area. There is no concrete cut-off to demarcate thedifference between micro- and macroarrays, and both types of arrays arecontemplated for use with the invention.

Microarrays are known in the art and generally consist of a surface towhich probes that correspond in sequence to gene products (e.g., cDNAs,mRNAs, oligonucleotides) are bound at known positions. In oneembodiment, the microarray is an array (e.g., a matrix) in which eachposition represents a discrete binding site for a product encoded by agene (e.g., a protein or RNA), and in which binding sites are presentfor products of most or almost all of the genes in the organism'sgenome. In certain embodiments, the binding site or site is a nucleicacid or nucleic acid analogue to which a particular cognate cDNA canspecifically hybridize. The nucleic acid or analogue of the binding sitemay be, e.g., a synthetic oligomer, a full-length cDNA, a less-than fulllength cDNA, or a gene fragment.

Although in certain embodiments the microarray contains binding sitesfor products of all or almost all genes in the target organism's genome,such comprehensiveness is not necessarily required. Usually themicroarray will have binding sites corresponding to at least 100, 500,1000, 4000 genes or more. In certain embodiments, arrays will haveanywhere from about 50, 60, 70, 80, 90, or even more than 95% of thegenes of a particular organism represented. The microarray typically hasbinding sites for genes relevant to testing and confirming a biologicalnetwork model of interest. Several exemplary human microarrays arepublicly available.

The probes to be affixed to the arrays are typically polynucleotides.These DNAs can be obtained by, e.g., polymerase chain reaction (PCR)amplification of gene segments from genomic DNA, cDNA (e.g., by RT-PCR),or cloned sequences. PCR primers are chosen, based on the known sequenceof the genes or cDNA, which result in amplification of unique fragments(e.g., fragments that do not share more than 10 bases of contiguousidentical sequence with any other fragment on the microarray). Computerprograms are useful in the design of primers with the requiredspecificity and optimal amplification properties. See, e.g., Oligo p1version 5.0 (National Biosciences). In an alternative embodiment, thebinding (hybridization) sites are made from plasmid or phage clones ofgenes, cDNAs (e.g., expressed sequence tags), or inserts therefrom(Nguyen et al., 1995, Genomics 29:207-209).

A number of methods are known in the art for affixing the nucleic acidsor analogues to a solid support that makes up the array (Schena et al.,1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; and Schena etal., 1995, Proc. Natl. Acad. Sci. USA 93:10539-11286).

Another method for making microarrays is by making high-densityoligonucleotide arrays (Fodor et al., 1991, Science 251:767-773; Peaseet al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; Lockhart et al.,1996, Nature Biotech 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and5,510,270; Blanchard et al., 1996, 11: 687-90).

Other methods for making microarrays, e.g., by masking (Maskos andSouthern, 1992, Nuc. Acids Res. 20:1679-1684), may also be used. Inprincipal, any type of array, for example, dot blots on a nylonhybridization membrane (see Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989), could be used, although, as will berecognized by those of skill in the art.

The nucleic acids to be contacted with the microarray may be prepared ina variety of ways, and may include nucleotides of the subject invention.Such nucleic acids are often labeled fluorescently. Nucleic acidhybridization and wash conditions are chosen so that the population oflabeled nucleic acids will specifically hybridize to appropriate,complementary nucleic acids affixed to the matrix. Non-specific bindingof the labeled nucleic acids to the array can be decreased by treatingthe array with a large quantity of non-specific DNA—a so-called“blocking” step.

When fluorescently labeled probes are used, the fluorescence emissionsat each site of a transcript array may be detected by scanning confocallaser microscopy. When two fluorophores are used, a separate scan, usingthe appropriate excitation line, is carried out for each of the twofluorophores used. Fluorescent microarray scanners are commerciallyavailable from Affymetrix, Packard BioChip Technologies, BioRobotics andmany other suppliers. Signals are recorded, quantitated and analyzedusing a variety of computer software.

According to the method of the invention, the relative abundance of anmRNA in two cells or cell lines is scored as a perturbation and itsmagnitude determined (i.e., the abundance is different in the twosources of mRNA tested), or as not perturbed (i.e., the relativeabundance is the same). As used herein, a difference between the twosources of RNA of at least a factor of about 25% (RNA from one source is25% more abundant in one source than the other source), more usuallyabout 50%, even more often by a factor of about 2 (twice as abundant), 3(three times as abundant) or 5 (five times as abundant) is scored as aperturbation. Present detection methods allow reliable detection ofdifference of an order of about 2-fold to about 5-fold, but moresensitive methods are expected to be developed.

In addition to identifying a perturbation as positive or negative, it isadvantageous to determine the magnitude of the perturbation. This can becarried out, as noted above, by calculating the ratio of the emission ofthe two fluorophores used for differential labeling, or by analogousmethods that will be readily apparent to those of skill in the art.

In certain embodiments, the data obtained from such experiments reflectsthe relative expression of each gene represented in the microarray.Expression levels in different samples and conditions may now becompared using a variety of statistical methods.

In certain embodiments, the cell comprises a tissue sample, which may bepresent on a tissue microarray. For example, paraffin-embeddedformalin-fixed specimens may be prepared, and punch “biopsy” cores takenfrom separate areas of the specimens. Each core may be arrayed into aseparate recipient block, and sections cut and processed as previouslydescribed, for example, in Konenen, J. et al., Tissue microarrays forhigh-throughput molecular profiling of tumor specimens, (1987) Nat. Med.4:844-7 and Chung, G. G. et al., Clin. Cancer Res. (In Press).

In other embodiments, the cell comprises a cell culture pellet, whichmay be present on a cell culture pellet microarray.

In certain embodiments, it is sufficient to determine the expression ofone or only a few genes, as opposed to hundreds or thousands of genes.Although microarrays may be used in these embodiments, various othermethods of detection of gene expression are available. This sectiondescribes a few exemplary methods for detecting and quantifying mRNA orpolypeptide encoded thereby. Where the first step of the methodsincludes isolation of mRNA from cells, this step may be conducted asdescribed above. Labeling of one or more nucleic acids may be performedas described above.

In one embodiment, mRNA obtained from a sample is reverse transcribedinto a first cDNA strand and subjected to PCR, e.g., RT-PCR. Housekeeping genes, or other genes whose expression does not vary may be usedas internal controls and controls across experiments. Following the PCRreaction, the amplified products may be separated by electrophoresis anddetected. By using quantitative PCR, the level of amplified product willcorrelate with the level of RNA that was present in the sample. Theamplified samples may also be separated on an agarose or polyacrylamidegel, transferred onto a filter, and the filter hybridized with a probespecific for the gene of interest. Numerous samples may be analyzedsimultaneously by conducting parallel PCR amplification, e.g., bymultiplex PCR.

“Dot blot” hybridization has gained wide-spread use, and many versionswere developed (see, e.g., M. L. M. Anderson and B. D. Young, in NucleicAcid Hybridization-A Practical Approach, B. D. Hames and S. J. Higgins,Eds., IRL Press, Washington D.C., Chapter 4, pp. 73-111, 1985).

In another embodiment, mRNA levels is determined by dot blot analysisand related methods (see, e.g. G. A. Beltz et al., in Methods inEnzymology, Vol. 100, Part B, R. Wu, L. Grossmam, K. Moldave, Eds.,Academic Press, New York, Chapter 19, pp. 266-308, 1985). In oneembodiment, a specified amount of RNA extracted from cells is blotted(i.e., non-covalently bound) onto a filter, and the filter is hybridizedwith a probe of the gene of interest. Numerous RNA samples may beanalyzed simultaneously, since a blot may comprise multiple spots ofRNA. Hybridization is detected using a method that depends on the typeof label of the probe. In another dot blot method, one or more probesfor 4E are attached to a membrane, and the membrane is incubated withlabeled nucleic acids obtained from and optionally derived from RNA of acell or tissue of a subject. Such a dot blot is essentially an arraycomprising fewer probes than a microarray.

Another format, the so-called “sandwich” hybridization, involvescovalently attaching oligonucleotide probes to a solid support and usingthem to capture and detect multiple nucleic acid targets (see, e.g., M.Ranki et al. (1983) Gene, 21:77-85; A. M. Palva, et al, in UK PatentApplication GB 2156074A, Oct. 2, 1985; T. M. Ranki and H. E. Soderlundin U.S. Pat. No. 4,563,419, Jan. 7, 1986; A. D. B. Malcolm and J. A.Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y. Stabinsky, in U.S. Pat.No. 4,751,177, Jan. 14, 1988; T. H. Adams et al., in PCT WO 90/01564,Feb. 22, 1990; R. B. Wallace et al. (1979) Nucleic Acid Res. 6, 11:3543;and B. J. Connor et al. (1983)PNAS 80:278-282). Multiplex versions ofthese formats are called “reverse dot blots.”

mRNA levels may also be determined by Northern blots. Specific amountsof RNA are separated by gel electrophoresis and transferred onto afilter which is then hybridized with a probe corresponding to the geneof interest. This method, although more burdensome when numerous samplesand genes are to be analyzed provides the advantage of being veryaccurate.

Another method for high throughput analysis of gene expression is theserial analysis of gene expression (SAGE) technique, first described inVelculescu et al. (1995) Science 270, 484-487. Among the advantages ofSAGE is that it has the potential to provide detection of all genesexpressed in a given cell type, provides quantitative information aboutthe relative expression of such genes, permits ready comparison of geneexpression of genes in two cells, and yields sequence information thatmay be used to identify the detected genes. Thus far, SAGE methodologyhas proved itself to reliably detect expression of regulated andnonregulated genes in a variety of cell types (Velculescu et al. (1997)Cell 88, 243-251; Zhang et al. (1997) Science 276, 1268-1272 andVelculescu et al. (1999) Nat. Genet. 23, 387-388.

Techniques for producing and probing nucleic acids are furtherdescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York, Cold Spring Harbor Laboratory, 1989).

Alternatively, the level of expression of a 4E regulon component orother gene of interest is determined by in situ hybridization. In oneembodiment, a tissue sample is obtained from a subject, the tissuesample is sliced, and in situ hybridization is performed according tomethods known in the art, to determine the level of expression.

In other methods, the level of expression of a 4E regulon component orother gene of interest is detected by measuring the level of proteinencoded by the gene. This may be done, e.g., by immunoprecipitation,ELISA, or immunohistochemistry using an agent, e.g., an antibody, thatspecifically detects the protein encoded by the gene. Other techniquesinclude Western blot analysis. Immunoassays are commonly used toquantitate the levels of proteins in cell samples, and many otherimmunoassay techniques are known in the art. The invention is notlimited to a particular assay procedure, and therefore is intended toinclude both homogeneous and heterogeneous procedures. Exemplaryimmunoassays which may be conducted according to the invention includefluorescence polarization immunoassay (FPIA), fluorescence immunoassay(FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay(NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay(RIA). An indicator moiety, or label group, may be attached to thesubject antibodies and is selected so as to meet the needs of varioususes of the method which are often dictated by the availability of assayequipment and compatible immunoassay procedures. General techniques tobe used in performing the various immunoassays noted above are known tothose of ordinary skill in the art.

In the case of polypeptides which are secreted from cells, the level ofexpression of these polypeptides may be measured in biological fluids.

The above-described methods may be performed using cells grown in cellculture, or on cell or tissue specimens from a subject. Specimens may beobtained from an individual to be tested using either “invasive” or“non-invasive” sampling means. A sampling means is said to be “invasive”if it involves the collection of nucleic acids from within the skin ororgans of an animal (including, especially, a murine, a human, an ovine,an equine, a bovine, a porcine, a canine, or a feline animal). Examplesof invasive methods include blood collection, semen collection, needlebiopsy, pleural aspiration, umbilical cord biopsy, etc. Examples of suchmethods are discussed by Kim, C. H. et al. (1992) J. Virol.66:3879-3882; Biswas, B. et al. (1990) Annals NY Acad. Sci. 590:582-583;Biswas, B. et al. (1991) J. Clin. Microbiol. 29:2228-2233. It is alsopossible to obtain a cell sample from a subject, and then to enrich itin the desired cell type. For example, cells may be isolated from othercells using a variety of techniques, such as isolation with an antibodybinding to an epitope on the cell surface of the desired cell type.

In certain embodiments, a single cell is used in the analysis. It isalso possible to obtain cells from a subject and culture the cells invitro, such as to obtain a larger population of cells from which RNA maybe extracted. Methods for establishing cultures of non-transformedcells, i.e., primary cell cultures, are known in the art. When analyzingfrom tissue samples or cells from individuals, it may be important toprevent any further changes in gene expression after the tissue or cellshas been removed from the subject. Changes in expression levels areknown to change rapidly following perturbations, e.g., heat shock oractivation with lipopolysaccharide (LPS) or other reagents. In addition,the RNA and proteins in the tissue and cells may quickly becomedegraded. Accordingly, in a preferred embodiment, the cells obtainedfrom a subject are snap frozen as soon as possible.

H. Diagnostic and Prognostic Applications of the 4E Regulon Components

1. Diagnostic Methods Comprising the Use of a Biological Sample

In certain embodiments are provided methods for diagnosing, monitoring,prognosing or staging, or predicting the outcome of a disease wherein 4Eregulon activity is dysfunctional, or the likelihood of developing adisease wherein 4E regulon activity is dysfunctional, comprise detectingthe level of, phosphorylation state of, or activity of at least one 4Eregulon component in a biological sample of a subject. In an exemplaryembodiment, the level of or activity of the 4E regulon component may bedetermined in a urine, saliva, blood or plasma sample from a subject.

The methods may comprise detecting the level of, phosphorylation stateof, or activity of at least one 4E regulon component in a biologicalsample of a subject and comparing that level to a control. A nonspecificcontrol, for example, may be GADPH or actin levels or activity. Anydeviation from the control level, phosphorylation state of, or activityof the at least one 4E regulon component may be indicative of a subjectsuffering from a disease wherein 4E regulon activity is dysfunctional,having a particular stage of a disease wherein 4E regulon activity isdysfunctional, about to develop a disease wherein 4E regulon activity isdysfunctional, etc. The degree or severity of a disease wherein 4Eregulon activity is dysfunctional may be determined based on the degreeof deviation in the level of, phosphorylation state of, or activity ofthe at least one 4E regulon component in a subject as compared to acontrol. For example, a subject exhibiting a greater deviation in thelevel of, phosphorylation state of, or activity of the at least one 4Eregulon component as compared to a control may indicate that the subjectis more susceptible to, or suffering from a more severe case of, adisease wherein 4E regulon activity is dysfunctional.

In certain embodiments of the methods describing the use of 4E reguloncomponents for diagnostic, screening and monitoring applications, thelevel of expression of, level/amount of, phosphorylation state of, oractivity of at least one non-4E regulon component may be determined andcompared as well. For example, the at least one non-4E regulon componentmay be selected from the group consisting of: ER (ER (alpha) gi:62821793; ER (beta) gis: 94538327, 94538324, 94538323), PR (gi:110611913), EGFR (gi: 41327737), HER2/neu (gi: 54792097, 54792095), andTMPRSS2:ETS gene fusions (Rubin, M A and Chinnaiyan, A M (2006) LabInvest 86: 1099).

The level of, phosphorylation state of, or activity of the at least one4E regulon component may be determined using a method known in the art.In embodiments wherein the at least one 4E regulon component is aprotein or a peptide corresponding to a region of a protein, the levelof the protein or peptide may be evaluated directly. For example, thelevel of at least one 4E regulon component may be determined usingimmunoassays. The activity of at least one 4E regulon component may beevaluated using an assay specific for the activity of that at least one4E regulon component.

Other methods for detecting the level or activity of 4E reguloncomponents in a biological sample are described further in Section Jbelow.

The level of, phosphorylation state of, or activity of the at least one4E regulon component in a subject may be compared to a control eitherquantitatively or qualitatively. For example, a qualitative (orunitless) comparison may be carried out by determining whether the levelof, phosphorylation state of, or activity of the at least one 4E reguloncomponent in a subject is higher, lower, or about the same as a control.Optionally, a qualitative comparison may be used to estimate themagnitude of difference in the level of, phosphorylation state of, oractivity of the at least one 4E regulon component in a subject ascompared to a control, such as, for example, a 2-fold change, a 50%change, etc. For example, a quantitative comparison may be carried outby determining the quantity of at least one 4E regulon component in asubject as compared to the quantity in a control, wherein the quantityhas some form of units attached (such as, for example, mg of protein,volume of a spot/band in a gel, intensity of a spot on a phosphoimageror autoradiogram exposure, volume of a spot on a chromatography plate,etc.).

In another embodiment, the level of, phosphorylation state of, oractivity of at least one 4E regulon component in a biological sample ofa subject may be used to calculate the physiological concentration ofthe at least one 4E regulon component found in a subject. Thephysiological concentration of the at least one 4E regulon component ina subject may then optionally be compared to a control.

In certain embodiments, subjects may be screened for levels of,phosphorylation state of, or activity of at least one 4E reguloncomponent on a regular basis (or at regular intervals) for purposes ofdiagnosis of a disease wherein 4E regulon activity is dysfunctional,staging of a disease wherein 4E regulon activity is dysfunctional or tomonitor the stage or development of a disease wherein 4E regulonactivity is dysfunctional. In one embodiment, screening for levels of,phosphorylation states of, or activity of 4E regulon components may becarried out about once every month, once every 3 weeks, once every 2weeks, once every 10 days, once every week, or about once every 144,120, 96, 72, 48, 24, or 12 hours.

It may be desirable to monitor symptoms of a disease wherein 4E regulonactivity is dysfunctional in addition to monitoring the level of,phosphorylation state of, or activity of at least one 4E reguloncomponent in a subject.

2. Cell- and Tissue-Based Diagnostics

In certain embodiments, a method of evaluating the expression of atleast one 4E regulon component in a cell or tissue from a subject maycomprise determining in the cell the level of expression of at least one4E regulon component. In other embodiments, it may comprise determiningthe level of, phosphorylation state of, or activity of at least one 4Eregulon component protein or mRNA (i.e. a gene product).

Methods of evaluating gene expression and protein activity arewell-known in the art. Exemplary methods by which the expression of theat least one 4E regulon component gene or gene product or the activityof, level of or phosphorylation state of the at least one 4E reguloncomponents are further described in Section J below.

The above-described method may further comprise comparing the determinedlevel of expression of at least one 4E regulon component gene or geneproduct with at least one reference set of levels of expression of theat least one 4E regulon component gene or gene product, wherein thereference set indicates the state of the cell associated with theparticular level of expression of the at least one 4E regulon componentgene or gene product.

Comparison to a reference set or profile is particularly useful inapplications of the above-described methods, for example, when they areused in methods for diagnosing and prognosing and predicting the onsetof a disease wherein 4E regulon activity is dysfunctional in a subject,or for screening candidate therapeutics for their efficacy in treating adisease wherein 4E regulon activity is dysfunctional.

For example, a method for diagnosing, prognosing or staging, orpredicting the onset of a disease wherein 4E regulon activity isdysfunctional may comprise: (a) determining in a cell of a subject thelevel of expression of at least one 4E regulon component gene or geneproduct. The level of expression of at least one 4E regulon componentobtained thereby may further be compared to a reference set of thelevels of expression of the at least one 4E regulon component gene orgene product associated with various states of a disease wherein 4Eregulon activity is dysfunctional.

Comparison of the expression level of at least one 4E regulon componentgene or gene product with reference expression levels, e.g., expressionlevels in diseased cells of a subject having a disease wherein 4Eregulon activity is dysfunctional or in normal counterpart cells, ispreferably conducted using computer systems. In one embodiment,expression levels are obtained in two cells and these two sets ofexpression levels are introduced into a computer system for comparison.In a preferred embodiment, one set of expression levels is entered intoa computer system for comparison with values that are already present inthe computer system, or in computer-readable form that is then enteredinto the computer system.

In one embodiment, the invention provides computer readable forms of thegene expression profile data of the invention, or of valuescorresponding to the level of expression of at least one 4E reguloncomponent gene or gene product. The values may be, for example, mRNAexpression levels or AQUA™ scores. The values may also be mRNA levels,AQUA™ scores, or other measure of gene expression normalized relative toa reference gene whose expression is constant in numerous cells undernumerous conditions. In other embodiments, the values in the computerare ratios of, or differences between, normalized or non-normalizedlevels in different samples.

The gene expression profile data may be in the form of a table, such asan Excel table. The data may be alone, or it may be part of a largerdatabase, e.g., comprising other expression profiles. For example, theexpression profile data of the invention may be part of a publicdatabase. The computer readable form may be in a computer. In anotherembodiment, the invention provides a computer displaying the geneexpression profile data.

In one embodiment, the invention provides methods for determining thesimilarity between the level of expression of at least one 4E reguloncomponent gene or gene product in a first cell, e.g., a cell of asubject, and that in a second cell, comprising obtaining the level ofexpression of at least one 4E regulon component gene or gene product ina first cell and entering these values into a computer comprising adatabase including records comprising values corresponding to levels ofexpression of the at least one 4E regulon component gene or gene productin a second cell, and processor instructions, e.g., a user interface,capable of receiving a selection of one or more values for comparisonpurposes with data that is stored in the computer. The computer mayfurther comprise a means for converting the comparison data into adiagram or chart or other type of output.

In another embodiment, at least one value representing the expressionlevel of at least one 4E regulon component gene or gene product isentered into a computer system, comprising one or more databases withreference expression levels obtained from more than one cell. Forexample, a computer may comprise expression data of diseased and normalcells. Instructions are provided to the computer, and the computer iscapable of comparing the data entered with the data in the computer todetermine whether the data entered is more similar to that of a normalcell or of a diseased cell.

In another embodiment, the computer comprises values of expressionlevels in cells of subjects at different stages of a disease wherein 4Eregulon activity is dysfunctional and the computer is capable ofcomparing expression data entered into the computer with the datastored, and produce results indicating to which of the expressionprofiles in the computer, the one entered is most similar, such as todetermine the stage of a disease wherein 4E regulon activity isdysfunctional in the subject.

In yet another embodiment, the reference expression profiles in thecomputer are expression profiles from cells of one or more subjectshaving a disease wherein 4E regulon activity is dysfunctional, whichcells are treated in vivo or in vitro with a drug used for therapy of adisease wherein 4E regulon activity is dysfunctional. Upon entering ofexpression data of a cell of a subject treated in vitro or in vivo withthe drug, the computer is instructed to compare the data entered to thedata in the computer, and to provide results indicating whether theexpression data input into the computer are more similar to those of acell of a subject that is responsive to the drug or more similar tothose of a cell of a subject that is not responsive to the drug. Thus,the results indicate whether the subject is likely to respond to thetreatment with the drug or unlikely to respond to it.

In one embodiment, the invention provides systems comprising a means forreceiving gene expression data for one or a plurality of genes; a meansfor comparing the gene expression data from each of said one orplurality of genes to a common reference frame; and a means forpresenting the results of the comparison. A system may further comprisea means for clustering the data.

In another embodiment, the invention provides computer programs foranalyzing gene expression data comprising (a) a computer code thatreceives as input gene expression data for at least one gene and (b) acomputer code that compares said gene expression data from each gene toa common reference frame.

The invention also provides machine-readable or computer-readable mediaincluding program instructions for performing the following steps: (a)comparing at least one value corresponding to the expression level of atleast one 4E regulon component gene or gene product in a query cell witha database including records comprising reference expression orexpression profile data of one or more reference cells and an annotationof the type of cell; and (b) indicating to which cell the query cell ismost similar based on similarities of expression profiles. The referencecells may be cells from subjects at different stages of a diseasewherein 4E regulon activity is dysfunctional. The reference cells mayalso be cells from subjects responding or not responding to a particulardrug treatment and optionally incubated in vitro or in vivo with thedrug.

The reference cells may also be cells from subjects responding or notresponding to several different treatments, and the computer systemindicates a preferred treatment for the subject. Accordingly, theinvention provides methods for selecting a therapy for a patient havinga disease wherein 4E regulon activity is dysfunctional; the methodscomprising: (a) providing the level of expression of at least one 4Eregulon component gene or gene product in a diseased cell of thepatient; (b) providing a plurality of reference profiles, eachassociated with a therapy; and (c) selecting the reference profile mostsimilar to the subject expression profile, to thereby select a therapyfor said patient. In a preferred embodiment step (c) is performed by acomputer. The most similar reference profile may be selected by weighinga comparison value of the plurality using a weight value associated withthe corresponding expression data.

A computer readable medium may further comprise a pointer to adescriptor of a stage of a disease wherein 4E regulon activity isdysfunctional or to a treatment for a disease wherein 4E regulonactivity is dysfunctional.

In operation, the means for receiving gene expression data, the meansfor comparing the gene expression data, the means for presenting, themeans for normalizing, and the means for clustering within the contextof the systems of the present invention may involve a programmedcomputer with the respective functionalities described herein,implemented in hardware or hardware and software; a logic circuit orother component of a programmed computer that performs the operationsspecifically identified herein, dictated by a computer program; or acomputer memory encoded with executable instructions representing acomputer program that may cause a computer to function in the particularfashion described herein.

Those skilled in the art will understand that the systems and methods ofthe present invention may be applied to a variety of systems, includingIBM®-compatible personal computers running MS-DOS® or MicrosoftWindows®. In an exemplary implementation, expression profiles arecompared using a method described in U.S. Pat. No. 6,203,987. A userfirst loads expression profile data into the computer system. Genesetprofile definitions are loaded into the memory from the storage media orfrom a remote computer, preferably from a dynamic geneset databasesystem, through the network. Next the user causes execution ofprojection software which performs the steps of converting expressionprofile to projected expression profiles. The projected expressionprofiles are then displayed.

In yet another exemplary implementation, a user first leads a projectedprofile into the memory. The user then causes the loading of a referenceprofile into the memory. Next, the user causes the execution ofcomparison software which performs the steps of objectively comparingthe profiles.

Exemplary diagnostic tools and assays are set forth below, whichcomprise the above-described methodology.

In one embodiment, the invention provides methods for determiningwhether a subject has or is likely to develop a disease wherein 4Eregulon activity is dysfunctional, e.g., predicting the onset of adisease wherein 4E regulon activity is dysfunctional, comprisingdetermining the level of expression of at least one 4E regulon componentgene or gene product in a cell of the subject and comparing these levelsof expression with the levels of expression of the genes or geneproducts in a diseased cell of a subject known to have a disease wherein4E regulon activity is dysfunctional, such that a similar level ofexpression of the genes or gene products is indicative that the subjecthas or is likely to develop a disease wherein 4E regulon activity isdysfunctional or at least a symptom thereof. In a preferred embodiment,the cell is essentially of the same type as that which is diseased inthe subject.

In another embodiment the expression profiles of genes or gene productsin the panels of the invention may be used to confirm that a subject hasa specific type of a disease wherein 4E regulon activity isdysfunctional, and in particular, that the subject does not have arelated disease or disease with similar symptoms. This may be important,in particular, in designing an optimal therapeutic regimen for thesubject. It has been described in the art that expression profiles maybe used to distinguish one type of disease from a similar disease. Forexample, two subtypes of non-Hodgkin's lymphomas, one of which respondsto current therapeutic methods and the other one which does not, couldbe differentiated by investigating 17,856 genes in specimens of patientssuffering from diffuse large B-cell lymphoma (Alizadeh et al. Nature(2000) 405:503). Similarly, subtypes of cutaneous melanoma werepredicted based on profiling 8150 genes (Bittner et al. Nature (2000)406:536). In this case, features of the highly aggressive metastaticmelanomas could be recognized. Numerous other studies comparingexpression profiles of cancer cells and normal cells have beendescribed, including studies describing expression profilesdistinguishing between highly and less metastatic cancers and studiesdescribing new subtypes of diseases, e.g., new tumor types (see, e.g.,Perou et al. (1999) PNAS 96: 9212; Perou et al. (2000) Nature 606:747;Clark et al. (2000) Nature 406:532; Alon et al. (1999) PNAS 96:6745;Golub et al. (1999) Science 286:531). Such distinction is known in theart as “differential diagnosis”.

In yet another embodiment, the invention provides methods fordetermining the stage of a disease wherein 4E regulon activity isdysfunctional. It is thought that the level of expression of at leastone 4E regulon component gene or gene product changes with the stage ofthe disease. This could be confirmed, e.g., by analyzing the level ofexpression of the gene or gene product in subjects having a diseasewherein 4E regulon activity is dysfunctional at different stages, asdetermined by traditional methods. For example, the expression profileof a diseased cell in subjects at different stages of the disease may bedetermined as described herein. Then, to determine the stage of adisease wherein 4E regulon activity is dysfunctional in a subject, thelevel of expression of at least one 4E regulon component gene or geneproduct, which varies with the stage of the disease, is determined. Asimilar level of expression of at least one 4E regulon component betweenthat in a subject and that in a reference profile of a particular stageof the disease, indicates that the disease of the subject is at theparticular stage.

Similarly, the methods may be used to determine the stage of the diseasein a subject undergoing therapy, and thereby determine whether thetherapy is effective. Accordingly, in one embodiment, the level ofexpression of at least one 4E regulon component gene or gene product isdetermined in a subject before the treatment and several times duringthe treatment. For example, a sample of RNA may be obtained from thesubject before the beginning of the therapy and every 12, 24 or 72 hoursduring the therapy. Samples may also be analyzed one a week or once amonth. Changes in expression levels of genes or gene products over timeand relative to diseased cells and normal cells will indicate whetherthe therapy is effective.

In yet another embodiment, the invention provides methods fordetermining the likelihood of success of a particular therapy in asubject having a disease wherein 4E regulon activity is dysfunctional.In one embodiment, a subject is started on a particular therapy, and theeffectiveness of the therapy is determined, e.g., by determining thelevel of expression of at least one 4E regulon component gene or geneproduct in a cell of the subject. A normalization of the level ofexpression of the gene, i.e., a change in the expression level of thegene or gene product such that their level of expression resembles morethat of a non diseased cell, indicates that the treatment should beeffective in the subject.

Prediction of the outcome of a treatment in a subject may also beundertaken in vitro. In one embodiment, cells are obtained from asubject to be evaluated for responsiveness to the treatment, andincubated in vitro with the therapeutic drug. The level of expression ofat least one 4E regulon component gene or gene product is then measuredin the cells and these values are compared to the level of expression ofthe at least one 4E regulon component in a cell which is the normalcounterpart cell of a diseased cell. The level of expression may also becompared to that in a normal cell. The comparative analysis ispreferably conducted using a computer comprising a database ofexpression profiles as described above. A level of expression of atleast one 4E regulon component gene or gene product in the cells of thesubject after incubation with the drug that is similar to their level ofexpression in a normal cell and different from that in a diseased cellis indicative that it is likely that the subject will respond positivelyto a treatment with the drug. On the contrary, a level of expression ofat least one 4E regulon component gene or gene product in the cells ofthe subject after incubation with the drug that is similar to theirlevel of expression in a diseased cell and different from that in anormal cell is indicative that it is likely that the subject will notrespond positively to a treatment with the drug.

Since it is possible that a drug does not act directly on the diseasedcells, but is, e.g., metabolized, or acts on another cell which thensecretes a factor that will effect the diseased cells, the above assaymay also be conducted in a tissue sample of a subject, which containscells other than the diseased cells. For example, a tissue samplecomprising diseased cells is obtained from a subject; the tissue sampleis incubated with the potential drug; optionally one or more diseasedcells are isolated from the tissue sample, e.g., by microdissection orLaser Capture Microdissection (LCM, see infra); and the expression levelof at least one 4E regulon component is examined.

The invention may also provide methods for selecting a therapy for adisease wherein 4E regulon activity is dysfunctional for a patient froma selection of several different treatments. Certain subjects having adisease wherein 4E regulon activity is dysfunctional may respond betterto one type of therapy than another type of therapy. In a preferredembodiment, the method comprises comparing the expression level of atleast one 4E regulon component gene or gene product in the patient withthat in cells of subjects treated in vitro or in vivo with one ofseveral therapeutic drugs, which subjects are responders or nonresponders to one of the therapeutic drugs, and identifying the cellwhich has the most similar level of expression of at least one 4Eregulon component to that of the patient, to thereby identify a therapyfor the patient. The method may further comprise administering thetherapy identified to the subject.

It will be appreciated by one of skill in the art that all of theafore-described methods may be modified to use the level or, activity ofor phosphorylation state of a 4E regulon component protein in the sameway as a gene expression level to achieve similar goals.

I. Methods of Identifying Therapeutics

1. Therapeutic Agent Screening

The present invention further relates to the use of at least one 4Eregulon component in methods of screening candidate therapeutic agentsfor use in treating a disease wherein 4E regulon activity isdysfunctional. The candidate therapeutics may be selected from thefollowing classes of compounds: nucleic acids, small molecules,polypeptides, proteins, peptidomimetics, or nucleic acid analogs. Insome embodiments, the candidate therapeutics may be in a library ofcompounds. These libraries may be generated using combinatorialsynthetic methods. In certain embodiments of the present invention, theability of said candidate therapeutics to bind a target protein may beevaluated by an in vitro assay. In certain embodiments, combinations ofcompounds or biologics may be screened for their effect on 4E reguloncomponent expression or activity to identify potential co-therapeuticsor combination therapies. For example, Ribavirin or analogs or prodrugsthereof may be screened along with interferon, GMCSF, GCSF, IL-12, IL-2,compounds that inhibit or down-regulated tyrosine kinase activity,chemotherapeutic agents such as platinum compounds and others describedin the definition above, biologics and cytotoxins etc. in addition tomeasurement of cell proliferation, cell division, and/or gene expressionas noted.

Biologics may include antibodies or antigen binding fragments thereof,that bind to a targetable component of a tumor cell, tumor vasculatureor tumor stroma. A “targetable component” of a tumor cell, tumorvasculature or tumor stroma, is preferably a surface-expressed,surface-accessible or surface-localized component, although componentsreleased from necrotic or otherwise damaged tumor cells or vascularendothelial cells may also be targeted, including cytosolic and/ornuclear tumor cell antigens.

Biologics may also include anti-tumor cell immunotoxins or coaguligandssuch as antibodies exemplified by the group consisting of B3 (ATCC HB10573), 260F9 (ATCC HB 8488), D612 (ATCC HB 9796) and KS1/4, said KS1/4antibody obtained from a cell comprising the vector pGKC23 10 (NRRLB-18356) or the vector pG2A52 (NRRL B-18357). Biologics may beanti-tumor stroma immunotoxins or coaguligands, for example, antibodiesthat bind to a connective tissue component, a basement membranecomponent or an activated platelet component; as exemplified by bindingto fibrin, RIBS or LIBS.

Biologics such as anti-tumor vasculature immunotoxins or coaguligandsmay also comprise ligands, antibodies, or fragments thereof, that bindto a surface-expressed, surface-accessible or surface-localizedcomponent of the blood transporting vessels, preferably the intratumoralblood vessels, of a vascularized tumor. Such antibodies include thosethat bind to surface-expressed components of intratumoral blood vesselsof a vascularized tumor, including aminophospholipids themselves, andintratumoral vasculature cell surface receptors, such as endoglin (TEC-4and TEC-11 antibodies), a TGF.beta. receptor, E-selectin, P-selectin,VCAM-1, ICAM-1, PSMA, a VEGF/VPF receptor, an FGF receptor, a TIE,.alpha.sub.v.beta.sub.3 integrin, pleiotropin, endosialin and MHC ClassII proteins. The antibodies may also bind to cytokine-inducible orcoagulant-inducible components of intratumoral blood vessels.

Other anti-tumor vasculature immunotoxins or coaguligands may compriseantibodies, or fragments thereof, that bind to a ligand or growth factorthat binds to an intratumoral vasculature cell surface receptor. Suchantibodies include those that bind to VEGF/VPF (GV39 and GV97antibodies), FGF, TGF.beta., a ligand that binds to a TIE, atumor-associated fibronectin isoform, scatter factor/hepatocyte growthfactor (HGF), platelet factor 4 (PF4), PDGF and TIMP. The antibodies, orfragments thereof, may also bind to a ligand:receptor complex or agrowth factor:receptor complex, but not to the ligand or growth factor,or to the receptor, when the ligand or growth factor or the receptor isnot in the ligand:receptor or growth factor:receptor complex.

Cytotoxic agents such as plant-, fungus- or bacteria-derived toxins(immunotoxins). Ricin A chain, deglycosylated ricin A chain, gelonin andangiopoietins may also be used in combination therapies.

In some embodiments, candidate therapeutic agents, or “therapeutics”,are evaluated for their ability to bind the at least one 4E reguloncomponent. In other embodiments, candidate therapeutics are evaluatedfor their ability to bind the at least one 4E regulon component gene orgene product. The ability of the candidate therapeutic to bind the geneor protein may be evaluated by an in vitro assay. In either embodiment,the binding assay may also be in vivo.

In still other embodiments, therapeutic agents targeting the at leastone 4E regulon component may be assessed by monitoring the symptoms of adisease wherein 4E regulon activity is dysfunctional in a subject,wherein the amelioration of or prevention of a disease wherein 4Eregulon activity is dysfunctional indicates the therapeutic agent may beuseful as a treatment.

The present invention further provides methods for evaluating candidatetherapeutic agents for their ability to modulate the expression of theat least one 4E regulon component gene by contacting the cells of asubject with said candidate therapeutic agents. In certain embodiments,the candidate therapeutic will be evaluated for its ability to normalizethe level of expression of the at least one 4E regulon component gene orgene product. In this embodiment, should the candidate therapeutic beable to normalize the gene expression so that a disease wherein 4Eregulon activity is dysfunctional is ameliorated, inhibited orprevented, it may be considered a candidate therapeutic for a diseasewherein 4E regulon activity is dysfunctional. The candidate therapeuticagents may be selected, for example, from the following classes ofcompounds: Ribavirin or ribavirin analogs, antisense nucleic acids,RNAi, small molecules, polypeptides, proteins, including antibodies,peptidomimetics, or nucleic acid analogs.

Alternatively, candidate therapeutic agents may be evaluated for theirability to inhibit the level of, phosphorylation state of, or activityof the at least one 4E regulon component protein by contacting the cellsof a subject with said candidate therapeutic agents. In certainembodiments, a candidate therapeutic may be evaluated for its ability toinhibit the level of, phosphorylation state of, or activity of the atleast one 4E regulon component. In this embodiment, a candidatetherapeutic agent that exhibits the ability to modulate the protein'sactivity may be considered a candidate therapeutic for treating adisease wherein 4E regulon activity is dysfunctional.

Furthermore, a candidate therapeutic may be evaluated for its ability tonormalize the level of turnover of a protein encoded by the at least one4E regulon component gene. In another embodiment, a candidatetherapeutic may be evaluated for its ability to normalize thetranslational level of a protein encoded by the at least one 4E reguloncomponent. In yet another embodiment, a candidate therapeutic may beevaluated for its ability to normalize the level of turnover of an mRNAencoded by the at least one 4E regulon component gene from the panels ofthe present invention.

In another embodiment of the invention, a drug is developed by rationaldrug design, i.e., it is designed or identified based on informationstored in computer readable form and analyzed by algorithms. More andmore databases of expression profiles are currently being established,numerous ones being publicly available. By screening such databases forthe description of drugs affecting the expression of the at least one 4Eregulon component gene in a manner similar to the change in geneexpression profile from a diseased cell to that of a normal cellcorresponding to the diseased cell, compounds may be identified whichnormalize gene expression in a diseased cell. Derivatives and analoguesof such compounds may then be synthesized to optimize the activity ofthe compound, and tested and optimized as described above.

2. Therapeutic Agent Screening Assays

Assays and methods of developing assays appropriate for use in themethods described above are well-known to those of skill in the art, andare contemplated for use as appropriate with the methods of the presentinvention. The ability of said candidate therapeutics to bind a targetmay be determined using a variety of appropriate assays known to thoseof skill in the art. In certain embodiments of the present invention,the ability of a candidate therapeutic to bind a target protein, othergene product or gene may be evaluated by an in vitro assay. In eitherembodiment, the binding assay may also be an in vivo assay. Assays maybe conducted to identify molecules that modulate the expression and oractivity of a gene or gene product. Alternatively, assays may beconducted to identify molecules that modulate the activity of a proteinencoded by a gene or gene product.

Examples of assays contemplated for use in the present inventioninclude, but are not limited to, competitive binding assay, directbinding assay, two-hybrid assay, cell proliferation assay, kinase assay,phosphatase assay, nuclear hormone translocator assay, fluorescenceactivated cell screening (FACS) assay, colony-forming/plaque assay, andpolymerase chain reaction assay. Such assays are well-known to one ofskill in the art and may be adapted to the methods of the presentinvention with no more than routine experimentation.

All of the above screening methods may be accomplished using a varietyof assay formats. In light of the present disclosure, those notexpressly described herein will nevertheless be known and comprehendedby one of ordinary skill in the art. The assays may identify drugs whichare, e.g., either agonists or antagonists, of expression of at least one4E regulon component gene or gene product or of a protein:protein orprotein-substrate interaction of at least one 4E regulon component, orof the role of at least one 4E regulon component gene product in thepathogenesis of normal or abnormal cellular physiology, proliferation,and/or differentiation and disorders related thereto. Assay formatswhich approximate such conditions as formation of protein complexes orprotein-nucleic acid complexes, enzymatic activity, and even specificsignaling pathways, may be generated in many different forms, andinclude but are not limited to assays based on cell-free systems, e.g.purified proteins or cell lysates, as well as cell-based assays whichutilize intact cells.

3. Therapeutic Agent Efficacy Screening

The efficacy of candidate therapeutics identified using the methods ofthe invention may be evaluated, for example, by a) contacting cells of asubject with a candidate therapeutic and b) determining its ability toameliorate, inhibit or prevent a disease wherein 4E regulon activity isdysfunctional or a symptom thereof in the subject. Alternatively, theefficacy of candidate therapeutics may be evaluated by comparing theexpression levels of at least one 4E regulon component gene or geneproduct in a cell of a subject having a disease wherein 4E regulonactivity is dysfunctional with that of a normal cell. In one embodiment,the expression level of the genes or gene products may be determinedusing microarrays or other methods of RNA quantitation, or by comparingthe gene expression profile of a cell treated with a candidatetherapeutic with the gene expression profile of a normal cell.

The efficacy of the compounds may then be tested in additional in vitroassays and in vivo, e.g. in animal studies. Expression of a 4E reguloncomponent may also be measured before and after administration of thetest compound to the animal. A normalization of the expression of a 4Eregulon component is indicative of the efficiency of the compound fortreating a disease wherein 4E regulon activity is dysfunctional in theanimal. Likewise the level of, phosphorylation state of, or activity ofa 4E regulon component may be measured before and after administrationof the test compound to the animal. A normalization of the level of,phosphorylation state of, or activity of a 4E regulon component isindicative of the efficiency of the compound for treating a diseasewherein 4E regulon activity is dysfunctional in the animal.

In certain embodiments, wherein the efficacy is tested in vivo, changes4E regulon component activity in response to a candidate therapeuticagent are monitored in PBMC. In other embodiments, fluid-based analysisof 4E regulon component levels, for example, VEGF, in response to acandidate therapeutic agent, are used.

J. Methods of Evaluating the Expression, Level or Activity of 4E RegulonComponent Genes and Proteins

The methods of diagnosing and prognosing a disease wherein 4E regulonactivity is dysfunctional by evaluating the level of expression and/orthe level of phosphorylation state of, or activity of at least one 4Eregulon component and methods of screening candidate therapeutic agentswhich modulate the expression and/or the level of, phosphorylation stateof, or activity of at least one 4E regulon component, described above,comprise determining the level of expression and/or the level of,phosphorylation state of, or activity of the at least one 4E reguloncomponent.

Methods for determining the expression level of a gene and the level of,phosphorylation state of, or activity of a gene or protein are wellknown in the art. For example, the expression level of a 4E reguloncomponent gene can be determined by reverse transcription-polymerasechain reaction (RT-PCR); dot blot analysis; Northern blot analysis andin situ hybridization. Alternatively, the level of a 4E reguloncomponent can be analyzed using an appropriate antibody. In certainembodiments, the amounts of a 4E regulon component is determined usingantibodies against the 4E regulon component.

In certain embodiments, the level of expression of a 4E reguloncomponent is determined by determining its AQUA™ score, e.g., by usingthe AQUA™ automated pathology system. AQUA™ (for Automated QuantitativeAnalysis) is a method of analysis of absolute measurement of proteinexpression in situ. This method allows measurements of proteinexpression within sub-cellular compartments that results in a numberdirectly proportional to the number of molecules expressed per unitarea. For example, to measure nuclear estrogen receptor (ER), the tissueis “masked” using keratin in one channel to normalize the area of tumorand to remove the stromal and other non-tumor material from analysis.Then an image is taken using DAPI to define a nuclear compartment. Thepixels within the mask and within the DAPI-defined compartment aredefined as nuclear. The intensity of expression of ER is then measuredusing a third channel. The intensity of that subset of pixels divided bythe number of pixels (to normalize the area from spot to spot) to givean AQUA™ score. This score is directly proportional to the number ofmolecules of ER per unit area of tumor, as assessed by a standard curveof cell lines with known levels of ER protein expression. This method,including details of out-of-focus light subtraction imaging methods, isdescribed in detail in a Nature Medicine paper (Camp, R. L., Chung, G.G. & Rimm, D. L. Automated subcellular localization and quantificationof protein expression in tissue microarrays. Nat Med 8, 1323-7 (2002)),as well as U.S. Ser. No. 10/062,308, filed Feb. 1, 2002, both of whichreferences are incorporated herein by their entireties.

In certain embodiments, a reporter gene assay is used to detect thelevel of expression of a 4E regulon component or to determine whether 4Eregulon component interactions are interrupted. Reporter systems thatmay be useful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in 4E activity, and binding assays known in theart, such as an two-hybrid or interaction trap assay (see also, U.S.Pat. No. 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al(1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al (1993) Oncogene 8:1693-1696), forsubsequently detecting agents which disrupt binding of the interactioncomponents to one another.

In certain embodiments, mass spectroscopy is used to evaluate levels ofprotein or phosphoryation states of protein. Protein characterization bymass spectroscopy first requires protein isolation followed by eitherchemical or enzymatic digestion of the protein into smaller peptidefragments, whereupon the peptide fragments may be analyzed by massspectrometry to obtain a peptide map. Liquid chromatography may be usedin conjunction with mass spectrometry. Mass spectrometry may also beused to identify post-translational modifications (e.g.,phosphorylation, etc.) of a polypeptide. Various mass spectrometers maybe used within the present invention. Representative examples include:triple quadrupole mass spectrometers, magnetic sector instruments(magnetic tandem mass spectrometer, JEOL, Peabody, Mass), ionspray massspectrometers (Bruins et al., Anal Chem. 59:2642-2647, 1987),electrospray mass spectrometers (including tandem, nano- andnano-electrospray tandem) (Fenn et al., Science 246:64-71, 1989), laserdesorption time-of-flight mass spectrometers (Karas and Hillenkamp,Anal. Chem. 60:2299-2301, 1988), and a Fourier Transform Ion CyclotronResonance Mass Spectrometer (Extrel Corp., Pittsburgh, Mass.).

Phosphorylation may be measured using any other method known in the art.Typically, methods of measuring phosphorylation are based on theradioactive detection method. In these methods, a sample containing theprotein of interest is incubated with activators and a substrate in thepresence of γ-³²P-ATP or γ-³²P-GTP. Often, a general and inexpensivesubstrate such as histone or casein is used. After a suitable incubationperiod, the reaction is stopped and the phosphorylated substrate (orprotein) is separated from free phosphate using gel electrophoresis orby binding the substrate or protein to a filter and washing to removeexcess radioactively-labeled free ATP. The amount of radio-labeledphosphate incorporated into the substrate or protein may measured byscintillation counting or by phosphorimager analysis. Alternatively,phosphorylation of a substrate or protein may be detected byimmunofluorescence using antibodies specific for a phosphoserine,phosphothreonine or phosphotyrosine residue (e.g., anti-phosphoserine,Sigma #P3430; anti-phosphothreonine, Sigma #P3555; andanti-phosphotyrosine, Sigma #P3300).

In other embodiments, methods of detecting the level of expression of a4E regulon component may comprise the use of a microarray. Arrays areoften divided into microarrays and macroarrays, where microarrays have amuch higher density of individual probe species per area. Microarraysmay have as many as 1000 or more different probes in a 1 cm² area. Thereis no concrete cut-off to demarcate the difference between micro- andmacroarrays, and both types of arrays are contemplated for use with theinvention.

Microarrays are known in the art and generally consist of a surface towhich probes that correspond in sequence to gene products (e.g., cDNAs,mRNAs, oligonucleotides) are bound at known positions. In oneembodiment, the microarray is an array (e.g., a matrix) in which eachposition represents a discrete binding site for a product encoded by agene (e.g., a protein or RNA), and in which binding sites are presentfor products of most or almost all of the genes in the organism'sgenome. In certain embodiments, the binding site or site is a nucleicacid or nucleic acid analogue to which a particular cognate cDNA canspecifically hybridize. The nucleic acid or analogue of the binding sitemay be, e.g., a synthetic oligomer, a full-length cDNA, a less-than fulllength cDNA, or a gene fragment.

Although in certain embodiments the microarray contains binding sitesfor products of all or almost all genes in the target organism's genome,such comprehensiveness is not necessarily required. Usually themicroarray will have binding sites corresponding to at least 100, 500,1000, 4000 genes or more. In certain embodiments, arrays will haveanywhere from about 50, 60, 70, 80, 90, or even more than 95% of thegenes of a particular organism represented. The microarray typically hasbinding sites for genes relevant to testing and confirming a biologicalnetwork model of interest. Several exemplary human microarrays arepublicly available.

The probes to be affixed to the arrays are typically polynucleotides.These DNAs can be obtained by, e.g., polymerase chain reaction (PCR)amplification of gene segments from genomic DNA, cDNA (e.g., by RT-PCR),or cloned sequences. PCR primers are chosen, based on the known sequenceof the genes or cDNA, which result in amplification of unique fragments(e.g., fragments that do not share more than 10 bases of contiguousidentical sequence with any other fragment on the microarray). Computerprograms are useful in the design of primers with the requiredspecificity and optimal amplification properties. See, e.g., Oligo p1version 5.0 (National Biosciences). In an alternative embodiment, thebinding (hybridization) sites are made from plasmid or phage clones ofgenes, cDNAs (e.g., expressed sequence tags), or inserts therefrom(Nguyen et al., 1995, Genomics 29:207-209).

A number of methods are known in the art for affixing the nucleic acidsor analogues to a solid support that makes up the array (Schena et al.,1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; and Schena etal., 1995, Proc. Natl. Acad. Sci. USA 93:10539-11286).

Another method for making microarrays is by making high-densityoligonucleotide arrays (Fodor et al., 1991, Science 251:767-773; Peaseet al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; Lockhart et al.,1996, Nature Biotech 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and5,510,270; Blanchard et al., 1996, 11: 687-90).

Other methods for making microarrays, e.g., by masking (Maskos andSouthern, 1992, Nuc. Acids Res. 20:1679-1684), may also be used. Inprincipal, any type of array, for example, dot blots on a nylonhybridization membrane (see Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989), could be used, although, as will berecognized by those of skill in the art.

The nucleic acids to be contacted with the microarray may be prepared ina variety of ways, and may include nucleotides of the subject invention.Such nucleic acids are often labeled fluorescently. Nucleic acidhybridization and wash conditions are chosen so that the population oflabeled nucleic acids will specifically hybridize to appropriate,complementary nucleic acids affixed to the matrix. Non-specific bindingof the labeled nucleic acids to the array can be decreased by treatingthe array with a large quantity of non-specific DNA—a so-called“blocking” step.

When fluorescently labeled probes are used, the fluorescence emissionsat each site of a transcript array may be detected by scanning confocallaser microscopy. When two fluorophores are used, a separate scan, usingthe appropriate excitation line, is carried out for each of the twofluorophores used. Fluorescent microarray scanners are commerciallyavailable from Affymetrix, Packard BioChip Technologies, BioRobotics andmany other suppliers. Signals are recorded, quantitated and analyzedusing a variety of computer software.

According to the method of the invention, the relative abundance of anmRNA in two cells or cell lines is scored as a perturbation and itsmagnitude determined (i.e., the abundance is different in the twosources of mRNA tested), or as not perturbed (i.e., the relativeabundance is the same). As used herein, a difference between the twosources of RNA of at least a factor of about 25% (RNA from one source is25% more abundant in one source than the other source), more usuallyabout 50%, even more often by a factor of about 2 (twice as abundant), 3(three times as abundant) or 5 (five times as abundant) is scored as aperturbation. Present detection methods allow reliable detection ofdifference of an order of about 2-fold to about 5-fold, but moresensitive methods are expected to be developed.

In addition to identifying a perturbation as positive or negative, it isadvantageous to determine the magnitude of the perturbation. This can becarried out, as noted above, by calculating the ratio of the emission ofthe two fluorophores used for differential labeling, or by analogousmethods that will be readily apparent to those of skill in the art.

In certain embodiments, the data obtained from such experiments reflectsthe relative expression of each gene represented in the microarray.Expression levels in different samples and conditions may now becompared using a variety of statistical methods.

In certain embodiments, the cell comprises a tissue sample, which may bepresent on a tissue microarray. For example, paraffin-embeddedformalin-fixed specimens may be prepared, and punch “biopsy” cores takenfrom separate areas of the specimens. Each core may be arrayed into aseparate recipient block, and sections cut and processed as previouslydescribed, for example, in Konenen, J. et al., Tissue microarrays forhigh-throughput molecular profiling of tumor specimens, (1987) Nat. Med.4:844-7 and Chung, G. G. et al., Clin. Cancer Res. (In Press).

In other embodiments, the cell comprises a cell culture pellet, whichmay be present on a cell culture pellet microarray.

In certain embodiments, it is sufficient to determine the expression ofone or only a few genes, as opposed to hundreds or thousands of genes.Although microarrays may be used in these embodiments, various othermethods of detection of gene expression are available. This sectiondescribes a few exemplary methods for detecting and quantifying mRNA orpolypeptide encoded thereby. Where the first step of the methodsincludes isolation of mRNA from cells, this step may be conducted asdescribed above. Labeling of one or more nucleic acids may be performedas described above.

In one embodiment, mRNA obtained from a sample is reverse transcribedinto a first cDNA strand and subjected to PCR, e.g., RT-PCR. Housekeeping genes, or other genes whose expression does not vary may be usedas internal controls and controls across experiments. Following the PCRreaction, the amplified products may be separated by electrophoresis anddetected. By using quantitative PCR, the level of amplified product willcorrelate with the level of RNA that was present in the sample. Theamplified samples may also be separated on an agarose or polyacrylamidegel, transferred onto a filter, and the filter hybridized with a probespecific for the gene of interest. Numerous samples may be analyzedsimultaneously by conducting parallel PCR amplification, e.g., bymultiplex PCR.

“Dot blot” hybridization has gained wide-spread use, and many versionswere developed (see, e.g., M. L. M. Anderson and B. D. Young, in NucleicAcid Hybridization-A Practical Approach, B. D. Hames and S. J. Higgins,Eds., IRL Press, Washington D.C., Chapter 4, pp. 73-111, 1985).

In another embodiment, mRNA levels is determined by dot blot analysisand related methods (see, e.g. G. A. Beltz et al., in Methods inEnzymology, Vol. 100, Part B, R. Wu, L. Grossmam, K. Moldave, Eds.,Academic Press, New York, Chapter 19, pp. 266-308, 1985). In oneembodiment, a specified amount of RNA extracted from cells is blotted(i.e., non-covalently bound) onto a filter, and the filter is hybridizedwith a probe of the gene of interest. Numerous RNA samples may beanalyzed simultaneously, since a blot may comprise multiple spots ofRNA. Hybridization is detected using a method that depends on the typeof label of the probe. In another dot blot method, one or more probesfor a 4E regulon component are attached to a membrane, and the membraneis incubated with labeled nucleic acids obtained from and optionallyderived from RNA of a cell or tissue of a subject. Such a dot blot isessentially an array comprising fewer probes than a microarray.

Another format, the so-called “sandwich” hybridization, involvescovalently attaching oligonucleotide probes to a solid support and usingthem to capture and detect multiple nucleic acid targets (see, e.g., M.Ranki et al. (1983) Gene, 21:77-85; A. M. Palva, et al, in UK PatentApplication GB 2156074A, Oct. 2, 1985; T. M. Ranki and H. E. Soderlundin U.S. Pat. No. 4,563,419, Jan. 7, 1986; A. D. B. Malcolm and J. A.Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y. Stabinsky, in U.S. Pat.No. 4,751,177, Jan. 14, 1988; T. H. Adams et al., in PCT WO 90/01564,Feb. 22, 1990; R. B. Wallace et al. (1979) Nucleic Acid Res. 6, 11:3543;and B. J. Connor et al. (1983) PNAS 80:278-282). Multiplex versions ofthese formats are called “reverse dot blots.”

mRNA levels may also be determined by Northern blots. Specific amountsof RNA are separated by gel electrophoresis and transferred onto afilter which is then hybridized with a probe corresponding to the geneof interest. This method, although more burdensome when numerous samplesand genes are to be analyzed provides the advantage of being veryaccurate.

Another method for high throughput analysis of gene expression is theserial analysis of gene expression (SAGE) technique, first described inVelculescu et al. (1995) Science 270, 484-487. Among the advantages ofSAGE is that it has the potential to provide detection of all genesexpressed in a given cell type, provides quantitative information aboutthe relative expression of such genes, permits ready comparison of geneexpression of genes in two cells, and yields sequence information thatmay be used to identify the detected genes. Thus far, SAGE methodologyhas proved itself to reliably detect expression of regulated andnonregulated genes in a variety of cell types (Velculescu et al. (1997)Cell 88, 243-251; Zhang et al. (1997) Science 276, 1268-1272 andVelculescu et al. (1999) Nat. Genet. 23, 387-388.

Techniques for producing and probing nucleic acids are furtherdescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York, Cold Spring Harbor Laboratory, 1989).

Alternatively, the level of expression of a 4E regulon component isdetermined by in situ hybridization. In one embodiment, a tissue sampleis obtained from a subject, the tissue sample is sliced, and in situhybridization is performed according to methods known in the art, todetermine the level of expression of the 4E regulon component.

In other methods, the level of expression of a 4E regulon component isdetected by measuring the level of protein encoded by the 4E reguloncomponent. This may be done, e.g., by immunoprecipitation, ELISA, orimmunohistochemistry using an agent, e.g., an antibody, thatspecifically detects the protein encoded by the gene. Other techniquesinclude Western blot analysis. Immunoassays are commonly used toquantitate the levels of proteins in cell samples, and many otherimmunoassay techniques are known in the art. The invention is notlimited to a particular assay procedure, and therefore is intended toinclude both homogeneous and heterogeneous procedures. Exemplaryimmunoassays which may be conducted according to the invention includefluorescence polarization immunoassay (FPIA), fluorescence immunoassay(FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay(NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay(RIA). An indicator moiety, or label group, may be attached to thesubject antibodies and is selected so as to meet the needs of varioususes of the method which are often dictated by the availability of assayequipment and compatible immunoassay procedures. General techniques tobe used in performing the various immunoassays noted above are known tothose of ordinary skill in the art.

In the case of polypeptides which are secreted from cells, the level ofexpression of these polypeptides may be measured in biological fluids.

The above-described methods may be performed using cells grown in cellculture, or on cell or tissue specimens from a subject. Specimens may beobtained from an individual to be tested using either “invasive” or“non-invasive” sampling means. A sampling means is said to be “invasive”if it involves the collection of nucleic acids from within the skin ororgans of an animal (including, especially, a murine, a human, an ovine,an equine, a bovine, a porcine, a canine, or a feline animal). Examplesof invasive methods include blood collection, semen collection, needlebiopsy, pleural aspiration, umbilical cord biopsy, etc. Examples of suchmethods are discussed by Kim, C. H. et al. (1992) J. Virol.66:3879-3882; Biswas, B. et al. (1990) Annals NY Acad. Sci. 590:582-583;Biswas, B. et al. (1991) J. Clin. Microbiol. 29:2228-2233. It is alsopossible to obtain a cell sample from a subject, and then to enrich itin the desired cell type. For example, cells may be isolated from othercells using a variety of techniques, such as isolation with an antibodybinding to an epitope on the cell surface of the desired cell type.

In certain embodiments, a single cell is used in the analysis. It isalso possible to obtain cells from a subject and culture the cells invitro, such as to obtain a larger population of cells from which RNA maybe extracted. Methods for establishing cultures of non-transformedcells, i.e., primary cell cultures, are known in the art.

When analyzing from tissue samples or cells from individuals, it may beimportant to prevent any further changes in gene expression after thetissue or cells has been removed from the subject. Changes in expressionlevels are known to change rapidly following perturbations, e.g., heatshock or activation with lipopolysaccharide (LPS) or other reagents. Inaddition, the RNA and proteins in the tissue and cells may quicklybecome degraded. Accordingly, in a preferred embodiment, the cellsobtained from a subject are snap frozen as soon as possible.

K. Agents that Bind 4E Regulon Components

Provided also are agents that bind 4E regulon components. Preferably,such agents are 4E regulon component antibodies or antigen-bindingfragments thereof, including polyclonal and monoclonal antibodies,prepared according to conventional methodology. Antibodies andantigen-binding fragments thereof that bind 4E regulon components areuseful for determining 4E regulon component levels.

Antibodies and antigen-binding fragments thereof that bind a 4E reguloncomponent and are useful for determining 4E regulon component levels,include but are not limited to: antibodies or antigen-binding fragmentsthereof that bind specifically to a 4E regulon component or fragments oranalogs thereof.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratrope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modem Immunology, Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′).sub.2 fragment, retains both of the antigen binding sites ofan intact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd Fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(Frs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, W. R. (1986) The Experimental Foundations of ModemImmunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) EssentialImmunology, 7th Ed., Blackwell Scientific Publications, Oxford). In boththe heavy chain Fd fragment and the light chain of IgG immunoglobulins,there are four framework regions (FR1 through FR4) separatedrespectively by three complementarity determining regions (CDR1 throughCDR3). The CDRs, and in particular the CDR3 regions, and moreparticularly the heavy chain CDR3, are largely responsible for antibodyspecificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762 and 5,859,205.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. Following immunization of these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (HAMA) responseswhen administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to 4E regulon component polypeptides and nucleicacids. These polypeptides may be derived also from sources other thanantibody technology. For example, such polypeptide binding agents can beprovided by degenerate peptide libraries which can be readily preparedin solution, in immobilized form or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind to 4Eregulon component molecules. This process can be repeated throughseveral cycles of reselection of phage that bind to the 4E reguloncomponent molecules. Repeated rounds lead to enrichment of phage bearingparticular sequences. DNA sequences analysis can be conducted toidentify the sequences of the expressed polypeptides. The minimal linearportion of the sequence that binds to the 4E regulon component moleculescan be determined. One can repeat the procedure using a biased librarycontaining inserts containing part of all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Yeast two-hybrid screening methods also may be used to identifypolypeptides that bind to the 4E regulon component molecules. Thus, 4Eregulon component molecules can be used to screen peptide libraries,including phage display libraries, to identify and select peptidebinding partners of the 4E regulon component molecules.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used for example to isolate and identify a 4E regulon component,e.g. to detect its expression in tissue samples. The antibodies may becoupled to specific diagnostic labeling agents for imaging of theprotein or fragment thereof. Exemplary labels include, but are notlimited to, labels which when fused to a 4E regulon component moleculeproduce a detectable fluorescent signal, including, for example, greenfluorescent protein (GFP), enhanced green fluorescent protein (EGFP),Renilla reniformis green fluorescent protein, GFPmut2, GFPuv4, enhancedyellow fluorescent protein (EYFP), enhanced cyan fluorescent protein(ECFP), enhanced blue fluorescent protein (EBFP), citrine and redfluorescent protein from discosoma (dsRED). In another embodiment, a 4Eregulon component polypeptide is conjugated to a fluorescent orchromogenic label. A wide variety of fluorescent labels are availablefrom and/or extensively described in the Handbook of Fluorescent Probesand Research Products 8^(th) Ed. (2001), available from MolecularProbes, Eugene, Oreg., as well as many other manufacturers.

In other embodiments, a 4E regulon component is fused to a molecule thatis readily detectable either by its presence or activity, including, butnot limited to, luciferase, fluorescent protein (e.g., green fluorescentprotein), chloramphenicol acetyl transferase, β-galactosidase, secretedplacental alkaline phosphatase, β-lactamase, human growth hormone, andother secreted enzyme reporters.

L. Kits

The present invention provides kits for practice of any of theaforedescribed methods. The present invention provides kits, for examplefor treating various cancers. For example, a kit may comprise one ormore pharmaceutical compositions (e.g. comprising compounds of FormulasI and II and/or gene therapy vectors) as described above and optionallyinstructions for their use. In still other embodiments, the inventionprovides kits comprising one or more pharmaceutical compositions and oneor more devices for accomplishing administration of such compositions.

In certain embodiments, kits may comprise antibodies against a 4Eregulon component. In other embodiments, a kit may comprise appropriatereagents for determining the level of protein activity in the cells of asubject.

In still other embodiments, a kit may comprise a microarray comprisingprobes of a 4E regulon component gene. A kit may comprise one or moreprobes or primers for detecting the expression level of a 4E reguloncomponent and/or a solid support on which probes are attached and whichmay be used for detecting expression of a 4E regulon component. A kitmay further comprise controls, buffers, and instructions for use.

Kits may also comprise a library of 4E regulon component gene expressionlevels associated with various cellular or disease states, e.g.,reference sets. The kits may be useful for identifying subjects that arepredisposed to developing a disease wherein 4E regulon activity isdysfunctional, as well as for identifying and validating therapeuticsfor a disease wherein 4E regulon activity is dysfunctional. In oneembodiment, the kit comprises a computer readable medium on which isstored one or more gene expression patterns associated with variouscellular or disease states, or at least values representing levels ofexpression of a 4E regulon component in various cellular or diseasestates. The kit may comprise expression profile analysis softwarecapable of being loaded into the memory of a computer system.

Kit components may be packaged for either manual or partially or whollyautomated practice of the foregoing methods. In other embodimentsinvolving kits, this invention contemplates a kit including compositionsof the present invention, and optionally instructions for their use.Such kits may have a variety of uses, including, for example, imaging,diagnosis, therapy, and other applications.

In addition to the embodiments, aspects and objects disclosed herein,including the claims appended hereto, the following paragraphs set forthadditional, non-limiting embodiments and other aspects of the presentinvention.

One aspect relates to a method for diagnosing a disease wherein 4Eregulon activity is dysfunctional, comprising: (a) determining in abiological sample from a subject the level of, phosphorylation state of,or activity of at least one 4E regulon component and (b) comparing thelevel of, phosphorylation state of, or activity of the at least one 4Eregulon component with the level of, phosphorylation state of, oractivity of the at least one 4E regulon component that is associatedwith a disease wherein 4E regulon activity is dysfunctional in a subjecthaving a disease wherein 4E regulon activity is dysfunctional, wherein asimilar level of, phosphorylation state of, or activity of the at leastone 4E regulon component is indicative that the subject has or is likelyto develop a disease wherein 4E regulon activity is dysfunctional or atleast a symptom thereof.

In some embodiments, the method for prognosing or staging a diseasewherein 4E regulon activity is dysfunctional, comprises: (a) determiningin a biological sample from a subject the level of, phosphorylationstate of, or activity of at least one 4E regulon component and (b)comparing the level of, phosphorylation state of, or activity of the atleast one 4E regulon component with the level of or activity of the atleast one 4E regulon component associated with a degree of, severity ofor stage of the disease wherein 4E regulon activity is dysfunctional ina subject, wherein a similar level of, phosphorylation state of, oractivity of the at least one 4E regulon component is indicative that thesubject has that degree of, severity of or stage of the disease wherein4E regulon activity is dysfunctional.

In some embodiments, the method for predicting the onset of a diseasewherein 4E regulon activity is dysfunctional, comprises: (a) determiningin a biological sample from a subject the level of, phosphorylationstate of, or activity of at least one 4E regulon component and (b)comparing the level of, phosphorylation state of, or activity of the atleast one 4E regulon component with the level of, phosphorylation stateof, or activity of the at least one 4E regulon component associated withthe likelihood of onset of the disease wherein 4E regulon activity isdysfunctional in a subject, wherein a similar level of, phosphorylationstate of, or activity of at least one 4E regulon component is indicativethat the subject has or is likely to develop the disease wherein 4Eregulon activity is dysfunctional or at least a symptom thereof.

In some embodiments, the sample is selected from the group consistingof: plasma, blood, urine, saliva and tissue.

In some embodiments, the disease wherein 4E regulon activity isdysfunctional is selected from the group consisting of: cellularhypertrophy, cancer, and ischemia reperfusion.

In some embodiments, the at least one 4E regulon component is selectedfrom the group consisting of: eIF4E; Cyclin D1; NBS/Nibrin; Pim-1;Cyclin B1; Cyclin A2; ODC; VEGF; Skp2; Cyclin E1; c-myc; FGF2; MMP-9;mdm2; caspase-9; bcl2; Bcl/xL; Fbox1; CGGbp1; P54nrb/NONO.1;Selenoprotein S; eIF4E-BP1; Akt1; PI3K; GSK3B; HuR; and mTOR/FRAP1.

In some embodiments, the at least one 4E regulon component is selectedfrom the group consisting of: 4E, NBS/Nibrin, Pim-1, VEGF, Cyclin D1,Cyclin A2, 4E-BP1, ODC and HuR.

In some embodiments, the level of, phosphorylation state of, or activityof at least two 4E regulon components is determined and compared.

In some embodiments, the level of, phosphorylation state of, or activityof at least one non-4E regulon component is determined and compared.

In some embodiments, the at least one non-4E regulon component isselected from the group consisting of: ER, PR, EGFR and Her2/neu.

In some embodiments, the level of the 4E regulon component is determinedby either mass spectrometry in combination with gas chromatography,HPLC, liquid chromatography or thin layer chromatography.

In some embodiments, the activity of the 4E regulon component isdetermined by an immunoassay or an assay specific for the activity ofthat 4E regulon component.

In some embodiments, the method for diagnosing a disease wherein 4Eregulon activity is dysfunctional, comprises: (a) determining in a cellof a subject the level of expression of at least one 4E reguloncomponent gene or gene product and (b) comparing the level of expressionof the at least one 4E regulon component gene or gene product with thelevel of expression of the at least one 4E regulon component gene orgene product associated with a disease wherein 4E regulon activity isdysfunctional in a subject having a disease wherein 4E regulon activityis dysfunctional, wherein a similar level of expression of the at leastone 4E regulon component gene or gene product is indicative that thesubject has or is likely to develop a disease wherein 4E regulonactivity is dysfunctional or at least a symptom thereof.

In some embodiments, a method for prognosing or staging a diseasewherein 4E regulon activity is dysfunctional, comprises: (a) determiningin a cell of a subject the level of expression of at least one 4Eregulon component gene or gene product and (b) comparing the level ofexpression of the at least one 4E regulon component gene or gene productwith the level of expression of theat least one 4E regulon componentgene or gene product associated with a degree of, severity of or stageof a disease wherein 4E regulon activity is dysfunctional in a subject,wherein a similar level of expression of the at least one 4E reguloncomponent gene or gene product is indicative that the subject has thatdegree of, severity of or stage of a disease wherein 4E regulon activityis dysfunctional.

In some embodiments, a method for predicting the onset of a diseasewherein 4E regulon activity is dysfunctional, comprises: (a) determiningin a cell of a subject the level of expression of at least one 4Eregulon component gene or gene product and (b) comparing the level ofexpression of the at least one 4E regulon component gene or gene productwith the level of expression of the at least one 4E regulon componentgene or gene product associated with the likelihood of onset of adisease wherein 4E regulon activity is dysfunctional in a subject,wherein a similar level of expression of the at least one 4E reguloncomponent gene or gene product is indicative that the subject has or islikely to develop a disease wherein 4E regulon activity is dysfunctionalor at least a symptom thereof.

In some embodiments, the disease wherein 4E regulon activity isdysfunctional is selected from the group consisting of: cellularhypertrophy, cancer, and ischemia reperfusion.

In some embodiments, the at least one 4E regulon component is selectedfrom the group consisting of: eIF4E; Cyclin D1; NBS % Nibrin; Pim-1;Cyclin B1; Cyclin A2; ODC; VEGF; Skp2; Cyclin E1; c-myc; FGF2; MMP-9;mdm2; caspase-9; bcl2; bcl/xL; Fbox1; CGGbp1; P54nrb/NONO.1;Selenoprotein S; eIF4E-BP1; Akt1; PI3K; GSK3B; HuR; and mTOR/FRAP1.

In some embodiments, the at least one 4E regulon component is selectedfrom the group consisting of: 4E, NBS/Nibrin, Pim-1, VEGF, Cyclin D1,Cyclin A2, eIF4E-BP1, ODC and HuR.

In some embodiments, the level of expression of at least two 4E reguloncomponent genes or gene products is determined and compared.

In some embodiments, the level of expression of at least one non-4Eregulon component gene or gene product is determined and compared.

In some embodiments, the at least one non-4E regulon component isselected from the group consisting of: ER, PR, EGFR, Her2/neu, and Etsoncogene.

In some embodiments, the level of expression of the at least one 4Eregulon component gene or gene product is determined by determining thelevel of expression of the at least one 4E regulon component gene.

In some embodiments, the level of expression of the at least one 4Eregulon component gene or gene product is determined by determining theamount of the at least one 4E regulon component gene or gene product inthe cell.

In some embodiments, the level of expression of the at least one 4Eregulon component gene or gene product is determined by determining theamount of the mRNA encoding the at least one 4E regulon component in thecell.

In some embodiments, the level of expression of the at least one 4Eregulon component gene or gene product is determined by determining theAQUA™ score of the at least one 4E regulon component gene or geneproduct.

In some embodiments, the AQUA™ score of the at least one 4E reguloncomponent gene or gene product is determined using the AQUA™ automatedpathology system.

In some embodiments, the level of expression of the at least one 4Eregulon component gene or gene product is determined using a microarray.

In some embodiments, the microarray comprises nucleic acids that areable to hybridize to the at least one 4E regulon component gene or geneproduct.

In some embodiments, the microarray comprises nucleic acids that areable to hybridize to a gene product of the at least one 4E reguloncomponent gene.

In some embodiments, the microarray comprises polypeptides that are ableto detect the at least one 4E regulon component gene or gene product.

In some embodiments, the cell comprises a tissue sample.

In some embodiments, the tissue sample is present on a microarray.

In some embodiments, the present invention relatest to an antibodyspecific for a 4E regulon component or a fragment thereof.

In some embodiments, a method for identifying at least one candidatetherapeutic for treating a disease wherein 4E regulon activity isdysfunctional comprises: (a) contacting a cell with at least onecandidate therapeutic, (b) determining in the cell pre- and post-contactwith the at least one candidate therapeutic the level of,phosphorylation state of, or activity of at least one 4E reguloncomponent, wherein modulation of the level of, phosphorylation state of,or activity of the at least one 4E regulon component indicates that atleast one candidate therapeutic may be a therapeutic agent for treatingor preventing the disease wherein 4E regulon activity is dysfunctional.

In some embodiments, a method for identifying at least one candidatetherapeutic for treating a disease wherein 4E regulon activity isdysfunctional comprises: (a) contacting a cell with at least onecandidate therapeutic, (b) determining in the cell pre- and post-contactwith the at least one candidate therapeutic the level of expression ofat least one 4E regulon component gene or gene product, whereinmodulation of the level of expression of the at least one 4E reguloncomponent gene or gene product indicates that at least one candidatetherapeutic may be a therapeutic agent for treating or preventing thedisease wherein 4E regulon activity is dysfunctional.

In some embodiments, a method for identifying at least one candidatetherapeutic for treating a disease wherein 4E regulon activity isdysfunctional comprises: (a) contacting at least one cell with at leastone candidate therapeutic, (b) determining in the at least one cell pre-and post-contact with the at least one candidate therapeutic whethercellular apoptosis has been restored, wherein restoration of cellularapoptosis indicates that the at least one candidate therapeutic may be atherapeutic agent for treating or preventing the disease wherein 4Eregulon activity is dysfunctional.

In some embodiments, the disease is a proliferative disease or cancer.

In some embodiments, a method for identifying at least one candidatetherapeutic for treating a disease wherein 4E regulon activity isdysfunctional comprises: (a) contacting at least one cell with at leastone candidate therapeutic, (b) determining in the at least one cell pre-and post-contact with the at least one candidate therapeutic whethercellular apoptosis has been inhibited, wherein inhibition of cellularapoptosis indicates that the at least one candidate therapeutic may be atherapeutic agent for treating or preventing the disease wherein 4Eregulon activity is dysfunctional.

In some embodiments, the disease is ischemia reperfusion injury

In some embodiments, the at least one candidate therapeutic is in alibrary of candidate therapeutics.

In some embodiments, the library is generated using combinatorialsynthetic methods.

In some embodiments, the two candidate therapeutics are evaluated andwherein one candidate therapeutic is a known therapeutic for a diseasewherein 4E regulon activity is dysfunctional.

In some embodiments, a method for selecting a therapy for a patienthaving a disease wherein 4E regulon activity is dysfunctional comprises:

providing at least one query value corresponding to the level ofexpression of at least one 4E regulon component gene or gene productwhose expression is characteristic of a disease wherein 4E regulonactivity is dysfunctional in a patient;

providing a plurality of sets of reference values corresponding tolevels of expression of at least at least one 4E regulon component geneor gene product whose expression is characteristic of a disease wherein4E regulon activity is dysfunctional in a patient, each reference valuebeing associated with a therapy; and

selecting the reference values most similar to the query values, tothereby select a therapy for said patient.

In some embodiments, selecting further includes weighing a comparisonvalue for the reference values using a weight value associated with eachreference values.

In some embodiments, the method comprises administering the therapy tothe patient.

In some embodiments, the query values and the sets of reference valuesare expression profiles.

In some embodiments, a method for selecting a therapy for a patienthaving a disease wherein 4E regulon activity is dysfunctional comprises:

providing at least one query value corresponding to the level of,phosphorylation state of, or activity of at least one 4E reguloncomponent whose expression is characteristic of a disease wherein 4Eregulon activity is dysfunctional in a patient;

providing a plurality of sets of reference values corresponding to thelevels of, phosphorylation states of, or activities of at least at leastone 4E regulon component whose expression is characteristic of a diseasewherein 4E regulon activity is dysfunctional in a patient, eachreference value being associated with a therapy; and

selecting the reference values most similar to the query values, tothereby select a therapy for said patient.

Also provided is a kit comprising reagents for the practice of any ofthe methods described herein and/or further comprising instructions foruse.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references including literature references, issued patents,published or non published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. (See, for example, Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I andII (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation(B. D. Hames & S. J. Higgins eds. 1984); (R. I. Freshney, Alan R. Liss,Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,A Practical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Vols. 154 and 155 (Wu et al. eds.), ImmunochemicalMethods In Cell And Molecular Biology (Mayer and Walker, eds., AcademicPress, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV(D. M. Weir and C. C. Blackwell, eds., 1986) (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

Example 1 Ribavirin Suppresses eIF4E-Mediated Oncogenic Transformationby Physical Mimicry of the 7-methyl Guanosine mRNA Cap

The eukaryotic translation initiation factor eIF4E is deregulated inmany human cancers, and its overexpression in cells leads to malignanttransformation. Oncogenic properties of eIF4E are directly linked to itsability to bind 7-methyl guanosine of the 5′ end of the mRNA. Here, weobserve that the antiviral guanosine analogue Ribavirin binds to eIF4Ewith micromolar affinity at the functional site used by 7-methylguanosine mRNA cap, competes with eIF4E:mRNA binding, and, at lowmicromolar concentrations, selectively disrupts eIF4E subcellularorganization and transport and translation of mRNAsposttranscriptionally regulated by eIF4E, thereby reducing levels ofoncogenes such as cyclin D1. Ribavirin potently suppresseseIF4E-mediated oncogenic transformation of murine cells in vitro, oftumor growth of a mouse model of eIF4E-dependent human squamous cellcarcinoma in vivo, and of colony formation of eIF4E-dependent acutemyelogenous leukemia cells derived from human patients. These findingsdescribe a specific, potent, and unforeseen mechanism of action ofRibavirin. Quantum mechanical and NMR structural studies offerdirections for the development of derivatives with improved cytostaticand antiviral properties. In all, Ribavirin's association with eIF4E mayprovide a pharmacologic means for the interruption ofpost-transcriptional networks of oncogenes that maintain and enhanceneoplasia and malignancy in human cancer.

General Methods

Reagents. All reagents were of ACS grade from Sigma-Aldrich except forTris-carboxyethyl phosphine (Pierce), Nonidet P-40 (ICN),isopropyl-β-D-thiogalactopyranoside (Lab Scientific), and Ribavirin(Calbiochem). Because of the presence of a highly toxic contaminant incommercially available Ribavirin (data not shown), Ribavirin and7-methyl guanosine (m⁷G) were purified by reverse-phase high-performanceliquid chromatography (Waters) using a semipreparative C₈ column (Vydac)and a linear acetonitrile gradient in 0.1% (vol/vol) aqueoustrifluoroacetic acid, lyophilized, and stored in a dessicator at −20° C.until use. This yielded purity of >99.99% as measured by usingthin-layer chromatography in 47:3 dichloromethane:methanol on aluminumoxide silica and electrospray ionization MS (data not shown). Rib4C(1-β-D-ribofuranosyl-1,2,3-triazole-4-carboxamide) was 99.98% pure and akind gift of Zhi Hong (ICN). Ribavirin-5′ triphosphate (RTP) wasobtained from Jena Bioscience.

Protein Expression. For fluorescence titrations, mouse eIF4E, whichdiffers from human eIF4E by four nonconserved amino acids, was producedas described. For NMR spectroscopy, mouse eIF4E was expressed as afusion with the B1 domain of protein G (G4E, kind gift of GerhardWagner, Harvard University, Cambridge, Mass.), in BL21 (DE-3) cells byinduction with 0.8 mM isopropyl-β-D-thiogalactopyranoside at 18° C. for20 h in M9 minimal media containing 1 g/l ¹⁵NH₄Cl as the sole nitrogensource (Cambridge Isotopes). Cells were lysed at 4° C. by sonication in0.1 M NaCl/50 mM Tris.HCl (pH 7.5/0.5 mM EDTA/0.5% (vol/vol) NonidetP-40/10 mM 2-mercaptoethanol/1 mM PMSF. Lysates were cleared bycentrifugation at 30,000×g, adsorbed onto m⁷G diphosphate-conjugatedagarose (Amersham Pharmacia), and extensively washed with 0.1 M NaCl/20mM Tris.HCl (pH 7.5)/0.5 mM EDTA (Buffer A) at 4° C. Subsequently, beadswere washed with 0.1 mM GTP in Buffer A, and G4E was eluted with 5 mMm⁷G in Buffer A. Eluate was diluted with 20 mM Na₂PO₄ (pH 7.5) to reduceNaCl concentration to 50 mM, applied to Sepharose Q anion exchangecolumn (Amersham Pharmacia), and eluted with a linear gradient of NaClin 20 mM Na₂PO₄ (pH 7.5) at 4° C. Eluate was dialyzed extensivelyagainst 0.1 M NaCl/50 mM Na₂PO₄ (pH 6.5)/5 mM DTT at 4° C. to produceapo-G4E, as verified by using NMR spectroscopy and fluorescencetitrations. Purity and identity of G4E were verified by using SDS/PAGEand electrospray ionization MS. Proteins were concentrated by usingAmicon concentrators (Millipore).

Fluorescence Spectroscopy. Fluorescence measurements were performed byusing a home-built fluorimeter, as described. All titrations wereperformed in 0.3 M NaCl/10 mM Na₂PO₄ (pH 7.5)/1 μM zinc in 0.3×0.3 cm²fluorescence cuvette (Hellma), by using eIF4E concentration of 2 μM.Collected emission spectra were integrated between 300 and 450 nm, andspectral contribution of eIF4E was determined by subtraction ofintrinsic fluorescence of added ligands by using extinction coefficientsof 740 and 970 M⁻¹cm⁻¹ at 295 nm for Ribavirin and Rib4C, respectively(data not shown), and corrected for the inner filter effect and for theminor attenuation of signal that occurs as a result of fluorophoredilution in the course of the titrations. Corrected relativefluorescence intensities were normalized, and fluorescence quenchingcurves were fit to a heuristic single-site-binding expression:I/I _(o) =K _(d) ^(n)/(x ^(n) +K _(d) ^(n))where x is ligand concentration, K_(d) is the apparent dissociationconstant, and n is the Hill coefficient.

m⁷G-Sepharose Affinity Chromatography. m⁷G-Sepharose beads (AmershamPharmacia, 20-μl slurry) were bound with 1.5 ml of 2 μM G4E, purified asdescribed above, in 0.3 M NaCl/0.1 M sodium phosphate (pH 7.5)/10 μMprotease-free BSA (USB)/0.1% Nonidet P-40 (Buffer B) for 30 min at roomtemperature. Beads were washed three times with Buffer B, and incubatedwith various concentrations of RTP or m⁷GTP in Buffer B for 30 min atroom temperature. Upon washing released G4E three times with Buffer B,G4E remaining bound to m⁷G-Sepharose was boiled in Laemmli buffer (10%glycerol/2% SDS/100 mM DTT/80 mM Tris.HCl/0.06% bromophenol blue, pH6.8), subjected to SDS/PAGE, and visualized by using Western blotting,as described below. Apparent inhibition constant was determined by usingK_(i)=IC₅₀·K_(d)/(P+K_(d)), where IC₅₀ is the apparent 50% inhibitorynucleotide concentration, P is effective protein concentration, andK_(d) is the apparent dissociation constant.

Cell Culture. NIH 3T3 mouse fibroblasts were maintained undersubconfluent conditions in DMEM (GIBCO/BRL)/10% (vol/vol) FBS/2 mMglutamate/0.1 mg/ml penicillin-streptomycin, at 37° C. in 5% CO₂. Forcell treatments, drugs were dissolved in PBS (pH 7.4) andfilter-sterilized. Untreated cells received filter-sterilized PBS.

Laser-Scanning Confocal Immunofluorescence Microscopy. Cells were washedwith PBS, fixed in methanol for 20 min at −20° C., and blocked in PBS,10% (vol/vol) FBS, and 0.1% (vol/vol) Tween 20 for 30 min at roomtemperature. Blocked cells were stained with primary antibodies againstNopp140 [1:50 (2)], Sc35 (1:50, Becton Dickinson), and eIF4E (1:50,Transduction Laboratories) in blocking solution for 3 h at roomtemperature. Upon washing with PBS, cells were stained with secondaryantibodies in blocking solution for 30 min: FITC-conjugated donkeyanti-rabbit antibody (Jackson ImmunoResearch), Texas red-conjugateddonkey anti-mouse antibody, and FITC-conjugated rabbit anti-mouseantibody, as appropriate. Subsequently, cells were washed with PBS andmounted in Vectashield supplemented with DAPI (Vector Laboratories).Fluorescence was observed by using 100× optical magnification and 2×digital zoom using Leica TCS-SP confocal microscope using excitationwavelengths of 488, 568, or 351/364 nm. All channels were detectedseparately, with no observable crosstalk. Micrographs represent singleoptical sections with a thickness of 300 nm, and are representative of100 cells.

Cell Fractionation. Cells were washed twice with PBS and lysed by slowpipetting in 0.14 M NaCl/10 mM Tris.HCl (pH 8.4)/1.5 mM MgCl₂/0.5%(vol/vol) Nonidet P-40/1 mM DTT/100 units/ml RNasin (Promega) at 4° C.Lysed suspensions were centrifuged at 1,000×g for 3 min at 4° C., andthe supernatant was saved as the cytoplasmic fraction. Nuclear pelletswere resuspended in lysis buffer, and 1/10 volume of 3.3% (wt/vol)sodium deoxycholate and 6.6% (vol/vol) Tween 40 was added under slowvortexing, and incubated at 4° C. for 5 min. Nuclei were sedimented bycentrifugation at 1,000×g for 3 min at 4° C., and the supernatant(postnuclear fraction) was added to the cytoplasmic fraction. Thisyielded intact nuclei, as observed by using light microscopy, with nosignificant cytoplasmic contamination, as evaluated using tRNA^(Lys) andβ-actin contents. Fractionated cytoplasm was free of nuclearcontamination, as indicated by absence of U6 snRNA and Sc35.

Northern Analysis. RNA from whole cells or nuclear and cytoplasmicfractions was extracted by using Trizol according to manufacturer'sinstructions (GIBCO). Isolated RNAs were treated with RNase-free DNase I(Promega) and 5-μg aliquots were resolved on 1% formaldehydeagarose geland transferred to a positively charged nylon membrane (Roche, Nutley,N.J.). Membranes were prehybridized in ULTRAhyb buffer (Ambion) andprobed with 20 pM cyclin D1 cDNA probe, 5 pM β-actin cDNA probe, 30 pMbiotinylated tRNA^(Lys) antisense oligo probe, and 30 pM biotinylated U6small nuclear RNA antisense oligo probe. cDNA probes were biotinylatedby using BrightStar psoralen-biotin and observed by using CDP Starchemiluminescence according to the manufacturer's instructions (Ambion).Band intensity and film response were quantified by using NIH IMAGE.

Western Analysis. Protein from whole cells or nuclear and cytoplasmicfractions was extracted by using 0.15 M NaCl/50 mM Tris.HCl (pH 7.4)/1%(vol/vol) Nonidet P-40/0.25% (wt/vol) sodium deoxycholate/1 mM EDTA/1 mMPMSF by incubating for 30 min at 4° C. Protein concentrations weredetermined by using bicinchoninic acid-copper reduction (Pierce) and20-μg aliquots were resolved using SDS/PAGE, transferred to Immobilon-Pmembrane (Millipore), blocked, and probed by using primary antibodiesagainst eIF4E (1:5000, Transduction Laboartories), cyclin D1 (1:500,Becton Dickinson), β-actin (1:5000, Sigma), c-myc (1:1000, BectonDickinson), and Sc35 (1:5000, Santa Cruz Biotechnology). Boundantibodies were chemiluminescently detected by using horseradishperoxidase-conjugated secondary antibodies (Amersham Pharmacia) andSuperSignal West Pico reagent according to manufacturer's instructions(Pierce).

Immunopurification of eIF4E and Semiquantitative RT-PCR. Nuclei isolatedfrom 3×10⁷ cells were suspended 0.3 M NaCl/50 mM Tris.HCl (pH 7.4)/0.05%(vol/vol) Nonidet P-40 (NET-2 buffer), mechanically disrupted by using amanual homogenizer, incubated for 1 h at 4° C., and sedimented bycentrifugation at 10,000×g. Soluble nuclear extracts were precleared byusing Sepharose-conjugated protein G (Amersham Pharmacia) for 30 min at4° C., incubated with 10 μg of mouse anti-eIF4E antibody (TransductionLaboartories) for 90 min at 4° C., and subsequently incubated overnightat 4° C. upon addition of 0.5 mg yeast tRNA (Sigma-Aldrich), 200units/ml RNasin (Ambion), and Sepharose-conjugated protein G. BoundSepharose was washed once with NET-2 buffer supplemented with 1 mg/mlheparin (Sigma-Aldrich) at 4° C., six times with NET-2 buffer alone, andsuspended in 100 mM Tris.HCl (pH 6.8)/4% (wt/vol) SDS/20% (vol/vol)glycerol/12% (vol/vol) 2-mercaptoethanol, and incubated for 5 min at 98°C. RNA was extracted once with 25:24:1 phenol:chloroform:isopropanol,twice with chloroform:isopropanol, precipitated overnight at −20° C.with 2.5 volumes of absolute ethanol, 0.1 volume of 5 M sodium acetate(pH 5.2), and 20 μg glycogen (Sigma-Aldrich), washed with 75% (vol/vol)ethanol, and resuspended in water. Messenger RNA of cyclin D1 wasamplified by using the ProStar first-strand RT-PCR system according tothe manufacturer's instructions (Stratagene), using forward5′-TCTACACTGACAACTCTATCCG-3′ (SEQ ID NO:2) and reverse5′-TAGCAGGAGAGGAAGTTGTTGG-3′ (SEQ ID NO: 3) primers. Although Ribavirindepletes the levels of eIF4E in the nucleus, eIF4E was still readilydetected in the IPs (data not shown), and importantly, the same amountsof mRNA were used for RT-PCR independent of the Ribavirin concentrationused. Thus, eIF4E-RNA binding from the nuclear fraction could beevaluated.

Purification of Ribosomes and Quantitative RT-PCR. Cell pellets (500 mg)were homogenized in 1 ml of ice cold lysis buffer (20 mM Hepes/10 mMmagnesium acetate/100 mM potassium acetate, pH 7.5) supplemented withprotease inhibitors (EDTA-free Complete, Roche) and 400 units/ml ofSUPERasine (Ambion), and incubated for 30 min on ice with occasionalvortexing. Lysates were sedimented at 3,000×g for 10 min at 4° C. topellet nuclei and cell debris. Supernatants were sedimented at 12,000×gfor 20 min at 4° C. to pellet mitochondria. Cleared supernatants werethen sedimented at 50,000 rpm (SW 50.1 rotor, Beckman) for 50 min at 4°C. to pellet ribosomes. Ribosomal pellets were resuspended in 200 μl ofice-cold lysis buffer and layered on top of 10-40% sucrose gradientbuffered with the lysis buffer, and centrifuged at 50,000 rpm for 80 minat 4° C. RNA content of fractions was ascertained by using the ratio ofabsorbance at 260:280 nm. RNA was isolated by using Trizol according tomanufacturer's instructions (GIBCO). RNA from each fraction wasquantified by spectrophotometry and 40 ng was converted into cDNA byusing the Sensiscript reverse transcription kit (Qiagen, Valencia,Calif.). Quantitative real-time PCR was carried out in triplicate byusing the QuantiTect SYBR green real-time PCR Kit (Qiagen) in an Opticonthermal cycler (MJ Research, Waltham, Mass.). The following genespecific primers were used: forward 5′-ACCACAGTCCATGCCATCAC-3′ (SEQ IDNO: 4) and reverse 5′-TCCACCACCCTGTTGCTGTA-3′ (GAPDH, SEQ ID NO: 5),forward 5′-CCTGACACCAATCTCCTCAACG-3′ (SEQ ID NO: 6) and reverse5′-TCTTCGCACTTCTGCTCCTCAC-3′ (cyclin D1, SEQ ID NO:7), forward5′-TGCCAAGTGGTCCCAGGCTG-3′ (SEQ ID NO: 8) and reverse5′-CGGCTTGAAGATGTACTCTAT-3′ (VEGF, SEQ ID NO: 9), and forward5′-GCATCAGCTTTCACGCTTG-3′ (SEQ ID NO: 10) and reverse5′-TCACCCACATGCATTTCAGG-3′ (ODC, SEQ ID NO: 11). Obtained real-time PCRprofiles were analyzed by using Opticon software (MJ Research).

Transformation Assay. Cells were transfected with 5 μg of pMV,pMV-eIF4E, or pMV-eIF4E mutants by using GeneJammer reagent according tomanufacturer's instructions (Stratagene), and selected using 1 mg/mlG418 sulfate for 48 h. Selected transfectants were plated at a densityof 20,000 cells per 100-mm² dish, and maintained in the presence of 1mg/ml G418 sulfate for 10 days. Dishes were washed with PBS, fixed withmethanol, and stained with Giemsa. Foci were counted manually andexperiments were repeated independently three times. Probability offocus formation is expressed as the number of foci, defined as havingreduced light refraction and being >50 cells, divided by 20,000 (per100-mm² dish).

Fluorescence-Activated Cell Scanning. For assessment of necrosis andapoptosis, cells were washed twice with PBS, suspended in 0.14 M NaCl/10mM Na-Hepes (pH 7.4/2.5 mM CaCl₂ at 4° C. at a density of 10⁶ cell perml, and stained with 5 μg/ml propidium iodide and FITC-conjugatedannexin V (Becton Dickinson) for 15 min at room temperature. Immediatelythereafter, cells were washed and analyzed by using a FACSCaliburfluorescence-activated cell scanner (Becton Dickinson). For assessmentof cell-cycle profiles, cells were washed twice with PBS, fixed, andpermeabilized in 70% (vol/vol) ethanol for 30 min at 4° C., andincubated in PBS containing 10 μg/ml propidium iodide and 30 units/mlRNase A at 37° C. for 30 min. For both measurements, detector gain andcompensation settings were adjusted to minimize autofluorescence ofunstained cells and channel crosstalk. For cell-cycle analysis, thepropidium iodide channel was gated based on light scattering to excludeclumped cells, which may artifactually skew the observed fluorescenceintensity.

Tetrazolium Dye Reduction. Cells were seeded at a density of 5,000 cellsper well and maintained in 100 μl per well in 96-well plates. Uponwarming the reagents to 37° C., 5 ml of sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT, Roche) dissolved at 1 mg/ml in RPMImedium 1640 without phenol red (GIBCO) was mixed with 0.1 ml of N-methyldibenzopyrazine methyl sulfate (PMS, Roche) dissolved at 0.38 mg/ml inPBS. Immediately after mixing, 50 μl of XTT-PBS solution was added toeach well, and cells were incubated for 2-4 h at 37° C. Production offormazan was quantified by using a μ Quant plate reader (Bio-TekInstruments) by monitoring the difference in absorbance at 492 and 690nm, as referenced to the difference in absorbance of samples containing50 μl of RPMI medium 1640 without phenol red. All experiments wererepeated three times.

Clonogenic Assay by Using Primary Human Leukemia Patient Cells. Acutemyelogenous leukemia (AML) M1, M5, and normal bone marrow specimens wereisolated from patients and processed as described. Primary AML cellswere obtained from peripheral blood of patients at the Markey CancerCenter, University of Kentucky Medical Center (Lexington, Ky.). Normalbone marrow was obtained as waste material after pathological analysis,surgical marrow harvest, or from the National Disease ResearchInterchange (Philadelphia). All tissues were obtained with the approvalof the Institutional Review Board and appropriate informed consent.Frozen CD34⁺ progenitor cells were thawed in Iscove's modifiedDulbecco's (IMD) medium supplemented with 10% (vol/vol) FBS. Viablecells were counted by using Trypan blue exclusion, and resuspended in 1%(vol/vol) H4100 methylcellulose IMD medium (Stem Cell Research),supplemented with 10% (vol/vol) BIT 9500 (Stem Cell Research), 2 mMglutamine (Sigma), 50 μg/ml low density lipoprotein (Sigma), and 50 μM2-mercaptoethanol. Cells were plated at a density of 2,000 viablecells/1.1 ml of medium per 35 mm dish, and cultured in the presence ofvarying concentrations of Ribavirin for 14 days. Colonies with >20 cellswere counted manually and experiments were repeated four times.

Mouse Model of Human Squamous Cell Carcinoma. Female 5- to 7-week-oldathymic NCr-nu/nu mice were obtained from Taconic Farms. Human FaDucells, derived from a hypopharyngeal squamous cell carcinoma, wereobtained from the American Type Culture Collection. Mice were inoculateds.c. into the right flank with 0.5×10⁶ cells in 50 μl PBS, and wererandomly segregated into two groups of 10 mice each. After 1 week ofengraftment, treatment was administered orally each day at a dose of 40μg/kg Ribavirin. Tumor size was ascertained by measuring tumor diameter,and statistical significance was ascertained by using a paired t test.

Calculation of Electrostatic Properties. Molecular geometries ofguanosine, m⁷G, Ribavirin(1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), Rib4C(1-β-D-ribofuranosyl-1,2,3-triazole-4-carboxamide), ICN3297(1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxylate), and tiazofurin(2-β-D-ribofuranosyl-4-thiazolecarboxamide) were generated by usingAMBER94, as implemented in INSIGHT 2000 (Accelrys, San Diego), optimizedby using Møller-Plesset (MP2) perturbation theory with the 6-31 G+(d)orbital basis set, and parameterized by point charge fitting in vacuum,as implemented in Gaussian 03 (Gaussian). Electrostatic potentials inaqueous solution were calculated by using the Poisson-Boltzmannapproximation with a dielectric constant of 80, as implemented in GRASP.

NMR Spectroscopy. ¹H, ¹⁵N heteronuclear single-quantum correlation(HSQC) spectra were recorded by using 500 MHz Bruker DRX spectrometer,in 0.1 M NaCl/50 mM Na₂PO₄ (pH 6.5)/5 mM DTT/5% (vol/vol) D₂O, at 288and 298 K, by using protein concentration of 0.8 mM. Backbone ¹H, ¹⁵Nresonances of G4E were obtained from ¹H, ¹³C, ¹⁵N resonance assignmentsof human eIF4E by direct spectral matching with a tolerance of 0.02 and0.2 ppm in the ¹H and ¹⁵N dimensions, respectively, leading toassignment of 64 unambiguous resonances in G4E, widely distributed inthe eIF4E structure. HSQC titrations were carried out by using m⁷G andRibavirin in 0.1 M NaCl/50 mM Na₂PO₄ (pH 6.5)/5 mM DTT/5% (vol/vol) D₂O,and ligand:protein ratios ranging from 0.3:1 to 5:1. Structuralparameters of Ribavirin binding were determined by using ¹⁵N-edited,¹⁵N-filtered, and double ¹⁵N-edited, filtered ¹H, ¹H NOESY spectroscopy.The contribution of spin diffusion to the observed nuclear Overhausereffect intensities was assessed by using mixing times ranging from 50 to250 msec, with no significant contribution of spin diffusion usingmixing time of 180 ms, as assessed from the linear dependence of nuclearOverhauser effect transfer on mixing time. Spectra were processed byusing NMRPIPE/NMRDRAW and analyzed by using NMRVIEW.

Results and Discussion

High-affinity binding of the m7G mRNA cap to mammalian eIF4E occurs byway of specific recognition of the methylated and consequentlypositively charged quaternary amine m7G base by two conservedtryptophans, W56 and W102, which form an aromatic stack as a result ofcation-pi and pi-pi interactions.

Binding of the uncharged tertiary amine guanosine to eIF4E is >5000-foldweaker. Because the pKa values of 1,2,4-triazoles are >12, making themprotonated and thus positively charged at physiological pH, weinvestigated whether eIF4E binds to the putatively cationic1,2,4-triazole-3-carboxamide of Ribavirin (FIG. 1). The affinity ofeIF4E for its nucleoside ligands in vitro can be measured by usingtryptophan fluorescence emission spectroscopy, whereby binding of ligandquenches fluorescence of tryptophans that stack with it. Ribavirin bindsto eIF4E with an apparent Kd of 8.4 uM, similar to that of m7Gnucleoside (FIGS. 1 a and 1 b). Mutation of one of the tryptophans,W56A, in the cap-binding site reduces affinity by 14-fold, whereasmutation of W73Aon the dorsal surface of eIF4E away from the cap-bindingsite has no significant effect on Ribavirin affinity (FIGS. 1 a and 1b). Similar results are obtained by using m7G. Furthermore, theRibavirin analogue 1-B-D-ribofuranosyl-1,2,3-triazole-4-carboxamide(Rib4C), which exhibits reduced antiviral and cellular effects andcontains an uncharged 1,2,3-triazole with a reduced pKa, fails to bindeIF4E. Because Ribavirin is nearly completely converted to RTP in cells,we measured the affinity of eIF4E for RTP. eIF4E binds RTP and m7GTPwith equal apparent dissociation constants of ca. 0.1 uM (FIGS. 1 a and1 b). By using m7G-Sepharose affinity chromatography, we observe thatRTP competes with eIF4E:m7G binding with an apparent inhibition constant(Ki) of ca. 0.3 uM, nearly indistinguishable from m7GTP itself (FIG. 1c). In all, these results indicate that Ribavirin binds eIF4E with highaffinity, at the functional site used by 5′ m7G mRNA cap, as a result ofcationic interaction with the cap-binding tryptophans, and suggest thatRibavirin competes with m7G 5′ mRNA cap binding to eIF4E in cells.

In mammalian cells, functions of eIF4E depend on its subcellularorganization. In the cytoplasm, eIF4E associates with ribosomes andfunctions in m7G cap-dependent mRNA translation. Up-regulation of eIF4Eincreases translation of only a specific set of sensitive mRNAs, thosethat are posttranscriptionally regulated by eIF4E at the level of mRNAtranslation. In the nucleus, eIF4E forms multiprotein structures, termedeIF4E nuclear bodies, and plays a role in nucleocytoplasmic mRNAtransport of a specific set of mRNA transcripts. The formation andfunction of these structures are linked with eIF4E's mRNA cap bindingbecause treatment of permeabilized cells with excess m7G cap analoguedisrupts eIF4E nuclear bodies but not other subnuclear structures.Consistently, disruption of eIF4E bodies impedes nucleocytoplasmiceIF4E-dependent mRNA transport.

Because Ribavirin binds eIF4E with high affinity and competes witheIF4E:m7G binding in vitro, we examined whether it affects subcellularorganization of eIF4E in cells. Thus, we treated NIH 3T3 fibroblastswith varying concentrations of Ribavirin for 48 h and monitored theirsubcellular organization by using immunofluorescence in conjunction withconfocal microscopy.

Ribavirin treatment has no apparent effects on chromatin structure(DAPI), organization of nucleoli and Cajal bodies (nucleolar proteinNopp140), structure of splicing speckles (Sc35 domains), and cellularmorphology (FIG. 2 a). In contrast, Ribavirin treatment disrupts eIF4Enuclear bodies, with this effect evident at 1 uM and nearly complete at10 uM (FIG. 2 a). To confirm this effect, we fractionated cells andexamined relative protein abundance in nuclear and cytoplasmic fractionsby using Western blotting methods. In agreement with the abovemicroscopy studies, Ribavirin treatment leads to redistribution of eIF4Ewithout affecting the distributions of predominantly nuclear Sc35 andcytoplasmic B-actin (FIG. 2 b). Importantly, Ribavirin treatment doesnot alter total protein levels of eIF4E but, rather, relocalizes themajority of the protein to the cytoplasm. Thus, Ribavirin may interferewith mRNA transport and translation of genes posttranscriptionallyregulated by eIF4E.

To test this possibility directly, we fractionated cells treated withRibavirin and assessed effects on nucleocytoplasmic mRNA transport bymonitoring cyclin D1 mRNA levels of nuclear and cytoplasmic fractions byusing subcellular fractionation and Northern methods or independently,using quantitative PCR. Ribavirin treatment impedes nucleocytoplasmictransport of cyclin D1 mRNA with an apparent EC50 of approx 1 uM, withnearly complete nuclear retention at 100 uM.

On the other hand, nucleocytoplasmic transport of B-actin and VEGF mRNAsis not affected even at 100 uM (FIGS. 3 a and 4 b), which is consistentwith insensitivity of their transport to eIF4E activity. Ribavirintreatment does not appear to affect splicing and 5′ capping of pre-mRNAsbecause co-transcriptional capping is required for pre-mRNA splicing,and both cyclin D1 and B-actin mRNAs are correctly spliced (FIG. 3 a).Moreover, Ribavirin does not appear to affect expression or localizationof nuclear RNAs with methylphosphate cap structures such as U smallnuclear RNAs, because the levels and distribution of U6 small nuclearRNA are not affected (FIG. 3 a). Similarly, Ribavirin treatment has noeffect on mRNA transcription and stability, because the totalsteady-state levels of cyclin D1, VEGF, and B-actin mRNAs are notaffected (FIGS. 3 b and 4).

We extended our studies to examine the effects of Ribavirin on mRNAtranslation in the cytoplasm by monitoring polysomal loading of mRNAstranslationally regulated by eIF4E. Polysomal fractions were prepared,and mRNA content was assessed by using real-time PCR. Ribavirintreatment has no significant effect on the polysomal loading profile ofcyclin D1 mRNA (FIG. 4 a), which is consistent with lack of regulationby eIF4E of cyclin D1 levels at the level of translation. In contrast,Ribavirin treatment leads to a shift of VEGF and ODC mRNAs from heavierpolysomal to lighter monosomal fractions, which have decreasedtranslational efficiency. The decrease of polysomal loadingis >1,000-fold (FIG. 4 a), in agreement with translational regulation ofVEGF and ODC levels by eIF4E. Thus, the apparent sensitivity of genes toRibavirin parallels their sensitivity to regulation by eIF4E, in termsof which genes are affected and the level(s) of regulation. Because manygenes are post-transcriptionally regulated by eIF4E, we focused oncyclin D1 as a model transcript because eIF4E's ability to modulate itsmRNA transport is well characterized.

Ribavirin treatment reduces levels of cyclin D1 protein with an apparentEC50 of 0.1-1 uM (FIG. 3 b), which is consistent with its inhibition ofnucleocytoplasmic cyclin D1 mRNA transport with an EC50 of approx. 1 uM(FIGS. 3 a and 4). In contrast, treatment with Rib4C, which does notbind to eIF4E in vitro (FIG. 1), cannot repress cyclin D1 proteinproduction in cells (FIG. 3 c). Furthermore, levels of B-actin and eIF4Eproteins, which are not post-transcriptionally regulated by eIF4E (FIG.3 a), are not reduced by Ribavirin treatment (FIG. 3 b). Thus,Ribavirin's specific interaction with eIF4E is required for Ribavirin'sability to suppress eIF4Edependent mRNA transport of cyclin D1.

We tested the ability of Ribavirin to directly alter the ability ofeIF4E to form ribonucleoproteins with transcripts sensitive toeIF4E-dependent mRNA transport (e.g., cyclin D1) and at the translationlevel (e.g., VEGF). Thus, we immunopurified eIF4E from nuclei ofRibavirin-treated cells and assessed its mRNA content by usingsemiquantitative RT-PCR (FIG. 3 d). Ribavirin treatment leads toinhibition of eIF4E binding to cyclin D1 mRNA in cells with an apparentEC50 of ca. 1 uM (FIG. 3 d), similar to the Kd for binding of itstriphosphate to eIF4E in vitro (FIG. 1) and to the EC50 for inhibitionof nucleocytoplasmic cyclin D1 mRNA transport and depletion of cyclin D1protein in cells (FIG. 3). Similarly, cytoplasmic eIF4E:VEGF mRNAcomplexes are partly abrogated, even by 1 uM Ribavirin (FIG. 3 d), whichis consistent with the observed alterations in polysomal loading byRibavirin (FIG. 4).

Importantly, cytoplasmic eIF4E:actin mRNA complexes are not disrupted,even at 100 uM Ribavirin, which is consistent with the insensitivity ofactin protein levels to Ribavirin. Ribavirin's effects are likely notlimited to eIF4E-mediated regulation of cyclin D1 mRNA transport andVEGF mRNA translation and include other genes regulatedposttranscriptionally by eIF4E.

eIF4E causes malignant transformation of cells when overexpressed.Mutagenesis studies indicate that its oncogenic properties are due, atleast in part, to deregulated transport of mRNAs of oncogenes and growthregulatory genes such as cyclin D1. Thus, we examined whether Ribavirintreatment and its inhibition of eIF4E-dependent mRNA transport andtranslation suppress eIF4E-mediated oncogenic transformation. Weoverexpressed eIF4E in NIH 3T3 cells and assayed transformation bymonitoring foci formation as a result of loss of contact growthinhibition. eIF4E levels in transfected cells are 10-fold greater thanendogenous levels in control cells (FIG. 5 a), leading to transformationand a significant increase in foci formation (FIG. 6 a). Overexpressionof eIF4E W56A cap-binding mutant fails to transform cells (FIG. 6 a), inagreement with earlier studies, even though it is expressed to similarlevels as wild-type eIF4E. Ribavirin suppresses eIF4E-mediatedtransformation with an apparent EC50 of 0.1-1 uM (FIGS. 3 a and 3 b). Incontrast, addition of Rib4C fails to reduce the number of foci formed,even at 100 uM (FIG. 3 a), which is consistent with its inability tobind eIF4E in vitro and inhibit eIF4E-mediated regulation of mRNAtransport and translation in cells (FIGS. 1 and 3 c). Observedsuppression of transformation is not due to nonspecific effects such asmetabolic toxicity or cell death (FIG. 5 b). Furthermore, low micromolarconcentrations of Ribavirin induce G1 cell-cycle arrest (FIG. 5 c),which is consistent with Ribavirin's down-regulation of cyclin D1 (FIG.3 b).

To examine the effect of Ribavirin on tumor growth in vivo, we obtainedspecimens of primary myeloid progenitor cells from patients with acutemyelogenous leukemias (AMLs) and comparable cells from normal bonemarrow. Previous studies indicated that a subset of AMLs have very highlevels of nuclear eIF4E and that cyclin D1 mRNA transport issubstantially upregulated in these cells. Reduction of nuclear eIF4Elevels led to a decrease in cyclin D1 mRNA transport to normal levels.Thus, we examined whether Ribavirin specifically alters growth of thissubset of AMLs. Isolated CD34+ progenitor cells were resuspended inmethylcellulose medium and cultured in the presence of variousconcentrations of Ribavirin for 14 days to assess their ability to formcolonies. Ribavirin potently repressed colony formation of primaryAML-M5 (French-American-British classification) progenitor cells with anapparent IC50 of ca. 1 uM (FIG. 6 b), which is consistent with theiroverexpression and dysregulation of eIF4E.

In contrast, similar concentrations of Ribavirin failed to represscolony formation of AML-M1 progenitor cells (FIG. 6 b), which isconsistent with non-up-regulated eIF4E levels and nondysregulated cyclinD1 mRNA transport in these cells. This tumor suppressive effect ofRibavirin at micromolar concentrations is distinct from its cellulartoxicity at millimolar concentrations (FIG. 5 b), as is evident from thelack of an effect on colony formation of normal bone marrow myeloidprogenitors at micromolar concentrations (FIG. 6 b). Treatment withRibavirin caused a marked suppression of tumor growth in a mouse modelof human squamous cell carcinoma (FIG. 5 c). We used FaDu cells derivedfrom a hypopharyngeal squamous cell carcinoma because they overexpresseIF4E, and form tumors in nude mice, as compared with nonmalignantepithelial cells.

Importantly, when levels of eIF4E are reduced to nonmalignant levels byusing antisense RNA, these cells are markedly less tumorigenic. Thus,nude mice were engrafted by using subcutaneous injection ofeIF4E-dependent FaDu cells and treated with 40 ug/kg Ribavirin orallyeach day, yielding a mean body concentration of ca. 1 uM. After 20 daysof Ribavirin treatment, mean tumor volume of animals in the treatmentgroup was 6-fold less than those in the untreated control group(P=0.023, n=10; FIG. 6 c). At this low concentration, Ribavirin wasapparently well tolerated and minimally toxic, as suggested by theabsence of treatment associated mortality and of effect on body weight(data not shown). Thus, Ribavirin's inhibition of eIF4E at lowmicromolar concentrations is correlated with inhibition ofeIF4E-mediated oncogenic transformation and tumor suppression in vitroand in vivo.

Binding of m7G mRNA cap by eIF4E is required for its nucleocytoplasmicmRNA transport, cytoplasmic translation, and oncogenic transformation.High-affinity binding of m7G cap by eIF4E is accomplished as a result ofspecific recognition of the cationic methylated base. Because Ribavirin,but not its neutral analogue, Rib4C, binds to eIF4E in vitro with thesame apparent affinity as the m7G cap and inhibits eIF4E's ability tobind mRNA and function in mRNA transport and translation in cells, weassessed the extent of similarity and molecular recognition by eIF4E ofRibavirin and m7G mRNA cap. Thus, we performed ¹H, ¹⁵N heteronuclearsingle-quantum correlation NMR spectroscopy (HSQC NMR) titrations ofeIF4E with m7G and Ribavirin. ¹H, ¹⁵N HSQC NMR spectroscopy reports onthe chemical environment of the individual ¹⁵NH amides of thepolypeptide backbone, thereby providing a sensitive probe of ligandbinding and accompanying conformational rearrangements.

In solution, eIF4E exists in low- and high-affinity conformations, theinterconversion of which is regulated by binding of partner proteinssuch as PML and ligands such as m7G mRNA cap, as observed by using CDspectroscopy titrations. Here, we observe a similar phenomenon upon theconversion from apo- to m7G-bound eIF4E by using HSQC NMR titrations,with the structuring or reorganization of 19 of 64 assigned residues of217 residues in eIF4E (FIG. 7 a), distributed throughout the structure(FIG. 7 c), in agreement with CD measurements. These residues includethe S7/S8 loop with W102, which stacks with the m7Gbase, and K106, whichcoordinates the ribose (FIG. 7 c). On the other hand, the S1/S2 loop ispreorganized in apo-eIF4E in the high-affinity conformation, with W56showing no significant changes in resonance intensity or chemical shiftupon cap binding (FIG. 7 a). Strikingly, conversion of apo-eIF4E toRibavirin-bound eIF4E involves an almost identical conformationalrearrangement, with little perturbation of the S1/S2 loop and W56 andsignificant structuring of the S7/S8 loop and W102 (FIG. 7 b), asindicated by nearly exact overlay of cap- and Ribavirin-bound spectra ofeIF4E (FIG. 7 b). These data are consistent with the reduced Ribavirinaffinity of W56A mutant and Ribavirin's ability to efficiently competewith m7G for binding to eIF4E (FIG. 1).

Double ¹⁵N-edited, filtered 1H, 1H NOESY spectra of nucleoside-saturatedeIF4E, which specifically identify ¹⁵NH groups of eIF4E in closeproximity (<5 Å) to nucleoside as a result of intermolecular NOEtransfer, are consistent with the binding sites of Ribavirin and m7Goverlapping (data not shown). Thus, eIF4E binds and recognizes Ribavirinin a manner similar to m7G cap, which is consistent with their similarbinding activities (FIG. 1). To assess the physical origin ofRibavirin's mimicry of m7G, we calculated electrostatic properties ofguanosine and Ribavirin analogues by using ab initio quantum mechanicaland continuum electrostatic methods. Only m7G and Ribavirin exhibitsignificant electropositive character in their aromatic rings (FIG. 8).Other nucleoside bases exhibit various degrees and patterns ofelectronegativity, including the inactive Ribavirin analogue Rib4C,which is not protonated and uncharged at neutral pH due to its1,2,3-triazole, inactive Ribavirin metabolite ICN3297, which is neutralbecause of its oxidized carboxamide (data not shown), guanosine analogueand inosine monophosphate dehydrogenase inhibitor tiazofurin, which isneutral due to its thiazole, and uncharged guanosine itself (FIG. 7).Thus, Ribavirin is a physical mimic of m7G.

There are two major cap-binding proteins in the cell, eIF4E and thecap-binding complex (CBC). Although both proteins intercalate m7Gbetween two aromatic residues, the affinity of the CBC for m7GpppG capis substantially higher (Kd ca. 10 nM), as compared with eIF4E (Kd ca.200 nM), because of more extensive interactions of the CBC with themethylated base, as well as with the adjacent pyrophosphate nucleotideas compared with eIF4E. Because Ribavirin's triazole ring would bemissing many of these additional contacts with the CBC and is missingthe adjacent base, Ribavirin interferes only with the functions ofeIF4E, and not those of the CBC, as observed here (FIG. 3).

Conclusions

Although widely studied, mechanisms of cellular action of Ribavirin andorigins of its antiviral effects remain enigmatic. Because of itssimilarity to guanosine, Ribavirin is suggested to inhibit 5′ mRNAcapping by competing with guanosine for guanylyl transferase, to inhibitguanosine biogenesis by mimicking guanosine for interaction with inosinemonophosphate dehydrogenase, and to be a mutagen by competing withguanosine for mRNA incorporation by RNA polymerases. Indeed, atmillimolar concentrations, such effects occur, leading to lethalmutagenesis of poliovirus (EC50 ca. 0.2 mM) and depletion of cellularguanosine pools (EC50 ca.0.1 mM), for example. Importantly, at lowmicromolar concentrations, Ribavirin does not appear to participate inguanosine metabolism, likely because of structural and energeticdifferences in m7G and/or guanosine-binding sites of involved proteins.Ribavirin does not appear to cause physiologic depletion of guanosinepools, as suggested by lack of metabolic toxicity (FIG. 5), and is notapparently mutagenic, as suggested by lack of cell death and unaffectedsynthesis and stability of produced proteins (FIGS. 3 and 5). Here, weobserve that Ribavirin inhibits the ability of eIF4E to promote mRNAtransport and translation of eIF4E-sensitive transcripts by antagonizingeIF4E:m7G mRNA cap binding and disrupting subcellular eIF4Eorganization. eIF4E overexpression does not increase protein synthesisglobally but, rather, affects the expression of a subset of transcriptsdefined as eIF4E-sensitive, including those studied here, such as cyclinD1, ODC, and VEGF.

Although the major point of this work was to elucidate a mechanism ofaction of Ribavirin and to characterize its potential anti-canceractivities, our findings have implications for mRNA translation as well.We show that selectivity of Ribavirin's inhibition of eIF4E stems fromthe selectivity of eIF4E's activity itself in terms of eIF4E'sposttranscriptional regulation of a specific set of eIF4E-sensitivetranscripts. Thus, just as eIF4E overexpression does not globallyincrease protein translation, Ribavirin is not a global inhibitor.Sensitivity to eIF4E appears to be inversely related to the complexityof UTRs of corresponding transcripts. Hence, Ribavirin-inducedinhibition of eIF4E specifically reduces translation of the transcriptsthat contain long and highly structured 5′ UTRs, including a number ofprotooncogenic mRNAs, e.g., VEGF, c-myc, and ODC. Conversely, Ribavirindoes not affect translational rates of housekeeping mRNAs, such asGAPDH, that bear short, unstructured 5′ UTRs. Electrostatic propertiesof guanosine-related nucleosides correlate directly with their point ofaction in cellular guanosine metabolism. For example, tiazofurin,despite having the same molecular geometry as Ribavirin, iselectronically similar to guanosine (FIG. 8) and, consequently, is apotent inosine monophosphate dehydrogenase inhibitor, binding to theguanosine allosteric effector site on inosine monophosphatedehydrogenase. Similarly, Rib4C is neutral (FIG. 8) and neither bindsnor inhibits eIF4E. On the other hand, Ribavirin is positively chargedat physiological pH because of its electronic structure (FIG. 8) and, asa result, antagonizes m7G mRNA cap binding by eIF4E. Ribavirin and itsderivatives offer a pharmacologic means to interrupt networks of tumorsuppressors and oncogenes that maintain and enhance neoplasia andmalignancy. For instance, deregulation of eIF4E leads to deregulation ofoncogenes such as cyclin D1 and myc, which, in turn, leads to furtherderegulation of eIF4E. eIF4E is a target of mitogenic stimulation and adirect transcriptional target of myc. Consistent with such selfreinforcing behavior, inactivation of myc leads to differentiation andsustained regression of tumors in a transgenic mouse model of osteogenicsarcoma. Similarly, antisense cyclin D1 reverts the phenotype of humancarcinoma cells toward normal and prevents tumor formation in mice.Complementarily, rapamycin suppresses chemoresistance in a mouselymphoma model, and this effect is reversed by dysregulation of eIF4E.Here, we demonstrate that a similar effect can be accomplishedpharmacologically by inhibiting eIF4E-dependent nucleocytoplasmic mRNAtransport and translation. It is becoming increasingly evident thatposttranscriptional regulation of gene expression plays a paramount rolein regulation of growth and development in eukaryotes, and disruption ofthis level of regulation contributes to a variety of human cancers.

Our findings indicate that Ribavirin acts in a previously unsuspectedmanner, at the level of post-transcriptional, eIF4E-mediated regulationof growth regulatory genes. It is likely that the apparent potency ofRibavirin's suppression of eIF4E-mediated oncogenic transformation invitro and in vivo involves down-regulation of a combination ofoncogenes, with cyclin D1 being a model transcript examined here.Further characterization of this unforeseen mechanism of Ribavirinaction and development of derivatives with improved antiviral andcytostatic properties are important directions for future work.

Example 2 eIF4E Specifically Binds to Ribavirin Using the Cap-BindingSite

The mechanism of action of Ribavirin (Virazole), a triazole carboxamideribonucleoside, has remained enigmatic since its discovery in the early1970s. Much of the confusion stemmed from its apparent activity againsta wide variety of seemingly unrelated viruses, as well as pleiotropiccellular effects dependent on concentration. Based on the similaritiesin the arrangement of hydrogen-bonding groups of Ribavirin andguanosine, Ribavirin was postulated to be a guanosine analog. Thisnotion is consistent with Ribavirin's effects at millimolarconcentrations on guanylyl transferases, inosine monophosphatedehydrogenase, and RNA-dependent RNA polymerases.

The prior Example sought to identify the nature of these specificeffects, and thereby help to define the specific mechanism of action ofRibavirin. Using ab initio quantum mechanics, we characterized thephysical properties of a variety of guanosine analogs, noting a strikingsimilarity in the electronic structure of Ribavirin and 7-methylguanosine. Using tryptophan emission fluorescence spectroscopy andnucleotide affinity chromatography, we measured the dissociation andinhibition constants of Ribavirin and the 7-methyl guanosine (m7G)binding protein eIF4E to be in the low micromolar range. Usingfluorescence microscopy, cell fractionation, Northern and Westernanalyses, and quantitative PCR, we observed Ribavirin's disruption ofsubcellular eIF4E localization, disruption of nuclear and cytoplasmiceIF4E:mRNA binding, inhibition of nucleocytoplasmic eIF4E-sensitive mRNAtransport, and inhibition of eIF4E-sensitive mRNA translation in livingcells, all at similar low micromolar concentrations. Using flowcytometry, colony formation, and tumor growth assays, we observedcytostatic and tumor-suppressive effects of Ribavirin in models ofeIF4E-dependent cancers in vitro and in vivo, as well as usingeIF4E-overexpressing leukemic blasts isolated from human patients, alsoat low micromolar concentrations. While Ribavirin exhibited physicalproperties similar to those of 7-methyl guanosine, its circularlypermuted chemical analog Rib4C did not, failing to bind eIF4E, toinhibit its functions in mRNA transport, translation, and tumorigenesis.This led us to conclude that Ribavirin is a physical mimic of the7-methyl guanosine mRNA cap.

In recent issues of RNA, Yan et al. (2005) and Westman et al. (2005)present findings that dispute this conclusion. Their results aretwofold: that Ribavirin does not bind to recombinant eIF4E in vitro, andthat Ribavirin does not inhibit cap-dependent translation of exogenousmRNAs in extracts prepared from cells. We are concerned that theseexperiments failed to elicit Ribavirin's effects and, similarly to theauthors, would like to discuss possible reasons for this. First, it iswell established that the binding of the m7G cap to eIF4E is highlydependent on solution conditions. Variations of several orders ofmagnitude (nanomolar to micromolar) can occur as a result of changes inionic strength, pH, and temperature. This likely depends on the physicalproperties and exact geometries of the cap binding site in the apo formof eIF4E under these conditions (data not shown), which themselves aredependent on the relative populations of various structural substatesthat apo-eIF4E is known to adopt in solution. Given that Ribavirin bindsthe cap-binding site of eIF4E, its apparent affinity for eIF4E wouldalso be condition dependent. Furthermore, given that Ribavirin'striazole carboxamide likely makes fewer atomic contacts with eIF4E ascompared to 7-methyl guanosine, Ribavirin's high-affinity binding toeIF4E would be expected to occur in a narrower range of solutionconditions.

With this in mind, we reproduced our original affinity chromatographyexperiment using an independent operator and new reagents side by sidewith the experiment of Yan et al. (2005). We reproduced experimentalconditions as published by us (0.3 M NaCl, 0.1 M sodium phosphate, 0.1%Nonidet P-40, 10 mM BSA at pH 7.5, room temperature), and thosedescribed by Yan et al. (2005) (0.1 M KCl, 10 mM HEPES-KOH, 0.2 mM EDTAat pH 8.0, presumed 4° C.). In agreement with our reported findings(Kentsis et al. 2004), micromolar concentrations of Ribavirintriphosphate (RTP) compete with the binding of eIF4E:m7G, similarly tothat of m7GTP itself (FIG. 9 a). In contrast, using the protocol of Yanet al. (2005) leads to an apparent failure of RTP to compete with m7Gbinding (FIG. 9 b). Thermodynamic meta-stability of eIF4E under varioussolution conditions is well described in the literature (Matsuo et al.1997; McGuire et al. 1998; Kentsis et al. 2001, 2004), leading toaggregation and linkage effects that can confound the apparent bindingof ligands (Fletcher and Wagner 1998; Cohen et al. 2001), particularlywhen using matrix-immobilized proteins, as in the experiments of Yan etal. (2005). In addition, the structure of apo-eIF4E is sensitive todifferences in pH between 7.5 and 8, as assessed by NMR chemical shiftperturbation (data not shown). Thus, the reported failure of Ribavirinto bind eIF4E in vitro by Yan et al. (2005) appears to be due, at leastin part, to the use of different solution conditions.

In contrast to Yan et al. (2005), who fail to observe Ribavirin'sbinding to eIF4E altogether, Westman et al. (2005) observe thatRibavirin binds to recombinant eIF4E in vitro, but does so with anaffinity two to four orders of magnitude lower than that measured by us(Kentsis et al. 2004; Westman et al. 2005). Measurements of ligandbinding using quenching of fluorescence emission often requirecorrections for the intrinsic fluorescence of added ligand and its innerfilter effect (Lakowicz 1999), neither of which appears to be consideredin our reading of Westman et al. (Niedzwiecka et al. 2002; Westman etal. 2005). While fluorescence quantum yields of nucleotides are lowerthan those of amino acids, they can be significant at concentrationsused in the above studies, potentially compensating for quenching ofprotein fluorescence upon binding, especially when Ribavirin's quenchingefficiency is twofold lower than that of 7-methyl guanosine because ofdifferences of the two compounds (Kentsis et al. 2001, 2004). Inaddition to this, titration of nucleotides leads to absorption ofincident and/or emitted light, potentially reducing the apparentemission of fluorescence. The lower extinction coefficient of Ribavirinas compared to 7-methyl guanosine (740 vs. 1600 M-1 cm-1 at 295 nm,respectively) may also contribute to the differences in apparentquenching (Kentsis et al. 2001, 2004).

Unfortunately, a direct methodological comparison is precluded by ourmonitoring of fluorescence emission of tryptophans including those thatdirectly bind 7-methyl guanosine (Kentsis et al. 2001, 2004), whileWestman et al. measured emission by both tryptophans and tyrosines(Niedzwiecka et al. 2002; Westman et al. 2005), which may bepreferentially quenched (excitation wavelengths of 295 nm and 280 nm,respectively). In addition, differences in solution conditions may alsocontribute to the observed differences in binding affinities. Assuggested by Westman et al. (2005), these methodological differences mayexplain the apparent differences in the measured affinities.

Nevertheless, in order to provide a decisive demonstration of binding ofRibavirin to eIF4E, one that does not involve indirect or ambiguousprobes of binding such as competition affinity chromatography andfluorescence quenching, we examined the binding of Ribavirin and eIF4Eby using electrospray mass spectrometry. A mixture of 20 mM purifiedrecombinant eIF4E and fourfold excess of both Ribavirin and GTP waselectrosprayed directly, and its mass/ionization spectrum was measured.The recorded spectrum is shown in FIG. 10 a and contains two sets ofmultiply charged ions, one with a population-weighted mean molecularmass of 31,402 Da, corresponding to apo-G4E (Zhou et al. 2001; Kentsiset al. 2004), and another of 31,649 Da (FIG. 10 b). This mass shift of247 Da is due to specific binding of Ribavirin (243 Da), and not of GTP(523 Da). In our published study, the specificity of Ribavirin's bindingto eIF4E was established using mutation of the cap-binding site W56A,which disrupts binding of Ribavirin, but not folding of the protein,similar to its disruption of binding of the 7-methyl guanosine cap(Kentsis et al. 2004).

Ribavirin's circularly permuted version Rib4C, which is chemicallyidentical but is not positively charged, failed to bind eIF4E. Andfinally, Ribavirin's binding leads to a similar conformationalrearrangement of eIF4E, as observed using NMR spectroscopy, as the oneinduced by the binding of 7-methyl guanosine cap, consistent withRibavirin's binding of the cap-binding site (Kentsis et al. 2004). Aligand-induced conformational change is also suggested by the cap-freecrystallographic structure reported by Volpon, et al. (2006) EMBO J.25(21):5138-49. Epub 2006 Oct. 12. Additional specificity controls aredescribed in Kentsis et al. (2004). Thus, eIF4E specifically binds toRibavirin using the cap-binding site, and experimental failure toobserve this interaction may be due to challenges of the particulartechniques used.

Another question examined by Yan et al. (2005) and Westman et al. (2005)concerns the effect of Ribavirin on eIF4E function. Both groups examinedfunctional effects in vitro, whereas we were concerned with Ribavirin'seffects in vivo (Kentsis et al. 2004). Cell extracts for translation ofexogenous mRNAs in vitro are well known for their unique properties,having altered compositions, stoichiometries, and activities, ascompared to those in living cells, where compartmentalization andmolecular organization are maintained and are of paramount importancefor a process as complex and regulated as mRNA translation. Althoughsuch extracts have been used with considerable success for the discoveryof translation factors, their significance for the characterization ofmechanisms of translation remains controversial. In this light,assessment of eIF4E activity by way of discrimination betweenefficiencies of translation of 7-methyl guanosine cap- and internalribosome binding site (IRES)-driven transcripts is problematic for anumber of reasons. The activity of each extract is optimized empiricallyin order to maximize the translational contribution of a particularfeature of an exogenous mRNA, a process that in no way guarantees theoverall mechanistic and functional fidelity that is absolutely requiredfor the characterization of novel activities such as that of Ribavirin.

For example, both Yan et al. (2005) and Westman et al. (2005) use cellextracts, albeit prepared from different cells and with differentmodifications, carefully optimized in order to maximize thetranslational synergy between the 50-cap and 30-poly(A) mRNA elements(Bergamini et al. 2000; Svitkin and Sonenberg 2004), a feature thatdepends neither solely nor specifically on the activity of eIF4E. Thisapparent synergy is due to the scaffolding activity of eIF4G, whichconcomitantly binds eIF4E, poly(A) binding proteins (PABPs), and theribosome (Michel et al. 2000), thereby coupling the affinities of eIF4Eand PABPs for the 50-cap and 30-poly(A) tail, respectively. Yet, thepresence of the poly(A) tail alone can also stimulate translation fromIRES in vitro (Svitkin et al. 2001), and eIF4E can recruit ribosomes inthe absence of cap binding (De Gregorio et al. 2001). Thus, in theexperiments of Westman et al. (2005), although competition with m7GpppGand m7GTP inhibits cap-driven translation at analog concentrations ofca. 0.1 mM while RpppG does not, specificity of this difference and itsmechanistic interpretation are indeterminate considering that theconcentration of m7 GpppG-capped transcript is ca. 1 nM (an excessof >100,000-fold) (Westman et al. 2005).

The interpretation of the findings of Yan et al. (2005) is even morecomplicated by their use of a bicistronic construct containing both the50-cap and an IRES that minimizes relative differences in efficiency asa result of competition for rate-limiting translation factors (Yan etal. 2005). Thus, treatment with 1 mM m7GDP leads to a reduction ofactivity of cap-driven firefly luciferase from ca. 8× to 2×10^5 lightunits: a fourfold effect, rather insignificant as compared to the molarexcess of cap analog to mRNA of >1,000,000-fold (mRNA concentration of 5mg/mL) (Yan et al. 2005). Considering that the concentration of eIF4E insimilarly used cell extracts is estimated to be ca. 400 nM (Rau et al.1996), the requirement of such high cap analog concentrations suggeststhat the examined process is not dependent strictly on eIF4E activityduring mRNA translation.

While we do not dispute that Ribavirin can be misincorporated into50-mRNA caps at millimolar concentrations, based on measurements ofviral production by Yan et al. (2005) and careful analysis of capstructures by Westman et al. (2005), we question the specificity of theobserved differences in translational efficiency between cap- andIRES-driven constructs in vitro, and their mechanistic interpretationwith respect to the mechanism of action of Ribavirin and our findings ofits inhibition of eIF4E sensitive translation in vivo. In this context,although Ribavirin failed to inhibit cap-dependent translation in vitroin the work of Yan et al. (2005) and Westman et al. (2005), this lack ofan effect may have to do with the lack of sensitivity of current cellextracts to eIF4E activity. In this regard, the distinction between cap-and eIF4Esensitive translation may be of paramount significance.Although the interaction of the 5′ 7-methyl guanosine cap with eIF4E isrequired for the translation of cap-dependent mRNAs, up-regulation ofeIF4E in cells does not increase levels of all proteins produced fromcap-dependent transcripts, but only of a specific subset includingcyclin D1 and VEGF, but not b-actin and GAPDH, for example (De Benedettiand Graff 2004). This effect occurs at the level of nucleocytoplasmictransport for some mRNAs, at the level of translation for others, andfor some at both (Rousseau et al. 1996). Thus, just as eIF4Eup-regulation does not globally increase cellular protein translation,Ribavirin is not a global inhibitor. Such specificity of Ribavirin'seffects on translation in cells was precisely observed in ourmeasurements of polysomal loading of mRNAs of cyclin D1, GAPDH, VEGF,and ODC (Kentsis et al. 2004).

In summary, just like that of the 7-methyl guanosine cap, Ribavirin'sbinding to eIF4E is dependent on solution conditions, but neverthelessoccurs robustly and specifically (FIGS. 9 and 10). To determine thephysiological relevance of interactions assessed in vitro, it isimportant to assess their functionality in vivo. Thus, we assessed thephysiological relevance of Ribavirin's binding to eIF4E in cells, inanimal models, as well as in tissues isolated from human patients(Kentsis et al. 2004). In all of these systems, Ribavirin antagonizedeIF4E functions in transport and translation of eIF4E-sensitive mRNAs atlow micromolar concentrations, similar to those at which it dissociatesfrom purified eIF4E in vitro. We hope that future collaborative workwill continue to define the specific mechanism and cellular effects ofthis rather simple chemically, but biologically complex, drug.

Example 3 Identification of a 100-nt Sequence From the Cyclin D1 3′UTRwhich Sensitizes this mRNA to eIF4E in the Nucleus and is Involved ineIF4E Mediated Cell Transformation

Abstract

The eukaryotic translation initiation factor eIF4E is a criticalmodulator of cellular growth with functions in the nucleus andcytoplasm. In the cytoplasm, recognition of the 5′ m7 G cap moiety onall mRNAs is sufficient for their functional interaction with eIF4E. Incontrast, we have shown that in the nucleus eIF4E associates andpromotes the nuclear export of cyclin D1, but not GAPDH or actin mRNAs.We determined that the basis of this discriminatory interaction is a100-nt sequence in the 3′ untranslated region (UTR) of cyclin D1 mRNA,we refer to as an eIF4E sensitivity element (4E-SE). We found thatcyclin D1 mRNA is enriched at eIF4E nuclear bodies, suggesting these arefunctional sites for organization of specific ribonucleoproteins. The4E-SE is required for eIF4E to efficiently transform cells, therebylinking recognition of this element to eIF4E mediated oncogenictransformation. Our studies demonstrate previously uncharacterizedfundamental differences in eIF4E-mRNA recognition between the nuclearand cytoplasmic compartments and further a novel level of regulation ofcellular proliferation.

Introduction

The eukaryotic translation initiation factor eIF4E is involved inmodulation of cellular growth. Moderate overexpression of eIF4E leads todysregulated growth and malignant transformation. The levels of eIF4Eare elevated in several human malignancies including a subset of myeloidleukemias and breast cancer. Importantly, both the nuclear andcytoplasmic functions of eIF4E contribute to its ability to transformcells. In the cytoplasm, eIF4E is required for cap-dependenttranslation, a process highly conserved from yeast to humans. Here,eIF4E binds the methyl 7-guanosine (m7G) cap moiety present on the 5′end of mRNAs and subsequently recruits the given mRNA to the ribosome.

In the nucleus, eIF4E functions to promote export from the nucleus tothe cytoplasm of at least two reported mRNAs, cyclin D1 and ornithinedecarboxylase (ODC), but does not alter GAPDH or actin mRNA export.Since the first report of the nuclear localization of eIF4E 12 yearsago, studies showed that up to 68% of cellular eIF4E is in the nucleus,where it associates with nuclear bodies in a wide variety of organismsincluding yeast, Drosophila, Xenopus, and humans. These bodies are foundin all cell types reported including nearly 30 cell lines and primarycells from diverse lineages such as NIH3T3, HEK293T, U2OS, K562, andU937. In mammalian cells, a large subset of eIF4E nuclear bodiescoincides with those associated with the promyelocytic leukemia proteinPML. PML was the first identified regulator of eIF4E-dependent mRNAexport. The RING domain of PML directly binds the dorsal surface ofeIF4E, reducing its affinity for the m7G cap by >100-fold. This loss ofcapbinding activity correlates with a loss of the mRNA export functionand loss of transformation activity.

There is evidence that the mRNA export function of eIF4E is linked toits oncogenic transformation activity. In a subset of primary humanmyeloid leukemia specimens, eIF4Edependent cyclin D1 mRNA export issubstantially up-regulated. Additionally, a mutant form of eIF4E, W73A,enters the nucleus colocalizing with endogenous eIF4E nuclear bodies,enhances the transport of cyclin D1 mRNAs to the cytoplasm andsubsequently transforms immortalized cells. This occurs despite the factthat W73A eIF4E cannot bind eIF4G and thus cannot act in translation.

Observations made by our group and the Sonenberg laboratory that eIF4Efunctionally discriminates between cyclin D1 and GAPDH mRNAs aresurprising because the traditional view is that eIF4E binds the m7G capfound on all mRNAs regardless of other sequence specific features. Thus,this functional discrimination presents a conundrum in terms of ourunderstanding of eIF4E mRNA recognition in the nucleus.

We explore the possibility that in the nucleus, eIF4E recognition ofmRNA is fundamentally different than in the cytoplasm. Here, we identifya 100-nt sequence from the cyclin D1 3′UTR which sensitizes this mRNA toeIF4E in the nucleus and is involved in eIF4E mediated celltransformation.

Materials and Methods

Constructs. All UTR-LacZ fusion constructs were made in pcDNA3.1LacZvector (Invitrogen) and positioned 5′ or 3′ of the coding region of LacZas appropriate. For cloning of cyclin D1 3′UTR, the NotI restrictionsite was created in pD1-1 construct (human cyclin D1 gene in pGEM7Zf),150 bp upstream of stop codon by in vitro mutagenesis (Quickchange kit;Stratagene) and full-length 3′UTR was cloned using NotI and XbaIdownstream of LacZ (referred here as LacZ-3′UTRFull). Fragmentscontaining the first part of cyclin D1 3′UTR were generated using NotIand EcoRI, and second part of cyclin D1 3′UTR using EcoRI and XbaI (notethat there is EcoRI site at the position 2,824 bp in human cyclin D1cDNA) and cloned under NotI-XbaI and EcoI-XbaI, downstream of LacZ (LacZ3′UTRA and LacZ 3′UTRB). Individual sequences were amplified usingspecific primers containing NotI or XbaI restriction sites at their 5′ends. LacZ

3′UTR2/3 contains segment 2,091-2,459 bp from cyclin D1 mRNA, LacZ 3′UTR3 contains sequence 2,361-2,459 bp, LacZ3/4 contains segment 2,361-2,565and LacZ 3′UTR4 contains sequence 2,481-2,565 bp from human cDNA. The5′UTR was amplified from cyclin D1 cDNA (ATCC MGC-23 16) and clonedusing the HindIII site, upstream of AUG codon for Xpress tag.pcDNA2Flag-eIF4E construct was made by inserting of eIF4E cDNA into theEcoRI-NotI sites (pcDNA2F vector was gift from Z. Ronai, BurnhamInstitute, La Jolla, Calif.). eIF4E mutants in pcDNA2Flag were made byin vitro mutagenesis (Quickchange kit; Stratagene). pMV vector,pMV-eIF4E wild type (a gift from N. Sonenberg, McGill University,Montreal, Quebec, Canada) or mutants, pLINKSV40-PML and bacterialexpression constructs were described previously. Human cyclin D1 cDNAwithout the full-length 3′UTR (ATCC MGC-2316) was cloned in pMV vectorusing EcoRI and HindIII (cyclin D1 truncated). Cyclin D1 full constructwas made by using HindIII-XbaI fragment from pcDNALacZ-3′TR that wasblunt ended and cloned under HindIII in pMV-cyclin D1Trunc (note thatthere is HindIII site in human cyclin D1 cDNA at position 1,206 bp).4E-SE-4 from cyclin D1 3′TR was PCR amplified, blunt ended and clonedunder HindIII in pMV-cyclin D1Trunc (cycD14E-SE).

Antibodies and Western analysis. Antibodies used against PML weredescribed previously (a gift from P. Freemont, Imperial College, London,UK and L. de Jong, University of Amsterdam, Amsterdam, Netherlands).Additional antibodies used include mouse monoclonal anti-eIF4E Ab (BDTransduction Laboratories), polyclonal anti-IF4E Ab (a gift from S.Morley, University of Sussex, Brighton, UK), mouse monoclonalanti-cyclin D1 Ab (BD Biosciences), mouse monoclonal anti-Xpress Ab(Invitrogen), mouse monoclonal anti-GAPDH antibody (MAB374; CHEMICONInternational, Inc.), anti-CBP80 pAb (a gift from L. Maquat, Universityof Rochester, Rochester, N.Y.; Ishigaki et al., 2001) anti-CBP-20 (agift from E. Izaurralde, EMBL, Heidelberg, Heidelberg, Germany). Westernanalysis was performed as described previously (Topisirovic et al.,2002, 2003).

Cell culture and transfection. NIH3T3, U2OS, HEK293T, and Nlog (a giftfrom H. Land, University of Rochester; cyclin D1−/− Perez-Roger et al.,1999) cells were maintained in 5% CO2 at 37C in DME (GIBCO BRL; LifeTechnologies), supplemented with 10% FBS, 100 U/ml penicillin, and 100U/ml streptomycin. eIF4E and PML stably transfected NIH3T3 were made asdescribed previously (Topisirovic et al., 2002, 2003a). Transienttransfection of NIH3T3 was performed using either GeneJammerTransfection Reagent (Stratagene) or Lipofectamine Plus reagent(Invitrogen) according to the manufacturer's instructions. Transienttransfections of HEK293T cells were performed using Calcium PhosphateTransfection kit (Invitrogen). Stable transfections of cyclin D1−/−cells were performed using Fugene 6 Transfection Reagent (Roche)according to the manufacturer's instructions. Anchorage-dependent fociformation assays were conducted as described previously (Cohen et al.,2001; Topisirovic et al., 2003a).

Immunopurification of eIF4E, isolation of RNA bound to eIF4E and RT-PCR.Nuclei were isolated from 3×10^9 HEK293T cells aliquoted appropriately,as previously described (Topisirovic et al., 2002), resuspended inice-cold NET-2 buffer (50 mM Tris-HCL, pH 7.4, 300 mM NaCl, 0.5%[vol/vol] NP-40, 1× complete protease inhibitors [Roche], 200 U/mlSUPERasein [Ambion]) and mechanically disrupted in dounce homogenizer(type B) on ice. Obtained nuclear extracts were cleared bycentrifugation at 16,000 g for 20 min at 4C. 1/20 of the supernatant wassplit in two and used to obtain nuclear RNA and protein, respectively.19/20 were split in three aliquots, two of which, when indicated in thetext, were incubated with 50 uM m7GpppG and 50 uM GpppG (NEB) in NET-2buffer for 30 min at 4° C. Each of the aforementioned aliquots was splitin two and immunoprecipitated as described previously (Ishigaki et al.,2001) with the following modifications: 10 u of anti-eIF4E mouse mAb(Transduction Laboratories) or 10 u of mouse IgG (Calbiochem) was usedper reaction and after immunoprecipitation, the beads were washed oncewith NET-2 buffer supplemented with 1 mg/ml of heparin (Sigma-Aldrich).Obtained RNA was treated with RNase free DNase (Promega) according tothe manufacturer's instruction. RNA was converted into cDNA using theSensiscript Reverse Transcription kit (QIAGEN). RT-PCR was performed intriplicate with the QuantiTect SYBR green RT-PCR Kit (QIAGEN) in Opticonthermal cycler (MJR). Obtained RT-PCR data was analyzed with Opticonsoftware (MJR). Primers used for cyclin D1 RT-PCR were cycF,5′CAGCGAGCAGCAGAGTCCGC-3′ (SEQ ID NO: 12) and cycr,5′-ACAGGAGCTGGTGTTCCATGGC-3′ (SEQ ID NO: 13); and for GAPDHamplification GAPDHF, 5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO: 14) andGAPDHR 5′-TCCACCACCCGTTGCTGTA-3′ (SEQ ID NO: 15). For RT-PCR methods,calculations were done as described by Applied Biosystems. Forsemi-quantitative PCR, 30 cycles were used, and for RT-PCR, standardmethods were used. Primers used for semi-quantitative amplification ofGAPDH were the same as for RT-PCR, and for cyclin D1 and actinamplification the following primers were used: cycHMF,5′-CACTTCCTCTCCAAAATGCCA-3′ (SEQ ID NO: 16); cycHMR,5′-CCTGGCGCAGGCTTGACTC-3′ (SEQ ID NO: 17); ActF,5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ (SEQ ID NO: 18); and ActR,5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′ (SEQ ID NO: 19).

Controls for quality of immunoprecipitation and fractionations. Severalsteps were taken to ensure that variability between experiments did notlead to false positive or false negative results. The immunoprecipitatedsample was tested to ensure that eIF4E immunoprecipitated itself andthat IgG did not bind eIF4E as determined by Western blotting. Thespecificity of the immuno-precipitation was determined using knownpositive and negative controls for eIF4E in the nuclear fraction. Thus,the ability of eIF4E antibodies to immunoprecipitate eIF4E but not CBP80(FIG. 11 g) or RNA Polymerase II was determined (Lai and Borden, 2000).These results are consistent with the findings from the Maquat and ourlaboratories where it was shown that the nuclear fraction of eIF4E doesnot associate with these proteins. Furthermore, positive controls forinteractions of eIF4E include the ability to associate with the PMLprotein (Cohen et al., 2001; Topisirovic et al., 2003a,b), as have beenreported numerous times. In addition, we demonstrate that thetransduction laboratory antibody against eIF4E used here colocalizeswith eIF4E antibodies produced in other laboratories (Topisirovic etal., 2004) indicating that the antibody is robust and reliable.Importantly, these experiments ensure that differences in association ofvarious mRNAs with eIF4E are NOT a result of differences inimmunoprecipitation efficiency or fractionation quality betweenexperiments.

For fractionation controls, the quality of each nuclear and cytoplasmicfraction was assessed by monitoring the subcellular distribution ofU6snRNA (nuclear) and tRNALys (cytoplasmic) as we reported previouslyand show throughout the text. Additional controls performed for eachfractionation include Western analysis of the splicing speckles proteinwhich served as a nuclear marker (Sc35) and B-actin, which served as acytoplasmic marker (Topisirovic et al., 2003 a,b). Additional fractioncontrols were done when sufficient material was available (Topisirovicet al., 2003 a,b). SNAAP protocol was performed as described previously(Trifillis et al., 1999) with the following modifications. Precleared250 ug of nuclear extracts were added to 50 ug of GST-protein beads in500 ul RBB buffer containing 0.5% NP-40, and after incubation of 30 minat 4C, 500 ug of yeast tRNA was added per reaction and incubatedovernight at 4° C. All washing of beads was performed in RBB buffercontaining 0.25% Triton X-100 and 0.5% NP-40.

Cellular fractionation and Northern analysis. Fractionation and RNAisolation were described previously (Lai and Borden, 2000; Topisirovicet al., 2002). For LacZ, PolyA RNA was purified from fractionated RNAusing Oligotex mRNA Mini Kit (QIAGEN). Probes for cyclin D1, GAPDH, U6,and tRNALys for Northern blot analysis were also previously described(Topisirovic et al., 2002). LacZ probe was made by PCR amplificationusing primers LacZF, 5′-CGGTCGCTACCATTACCAGTT-3′ (SEQ ID NO: 20) andLacZR, 5′-GACGTTGTAAAACGACGGGAT-3′ (SEQ ID NO: 21), and labeled usingBrightStar Psoralen-Biotin kit (Ambion).

Immunofluorescence, in situ hybridization, and laser scanning confocalmicroscopy. Immunofluorescence experiments were as described previously(Cohen et al., 2001; Topisirovic et al., 2002). Fluorescence wasobserved using 100× optical magnification and 2× digital zoom, unlessindicated otherwise, on an inverted laser scanning confocal microscope(model TCS-SP (UV); Leica) exciting at 488, 568, or 351/364 nm (at RT).All channels were detected separately, and no cross talk between thechannels was observed. Micrographs represent single sections through theplane of cells with a thickness of ca. 300 nm. Experiments were repeatedthree times with more than 500 cells in each sample. In situhybridization was performed according to Spector et al. (1998), usingnick-translated DIG-11-dUTP-labeled (Nick Translation Kit; Roche) cyclinD1 and GAPDH PCR-amplified fragments (cyclin D1 specific 5SA,5′-CATGGAACACCAGCTCCTGT-3′ (SEQ ID NO: 22) and 3SA,5′-CGCAGCCACCACGCTCCC-3′ (SEQ ID NO: 23); and GAPDH specific GAPDHHF,5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO: 24) and GAPDHMR,5′-TCCACCACCCTGTTGCTGGGG-3′ (SEQ ID NO: 25)) and detected using anti-DIGFab fragments (Roche) followed by donkey anti-sheep Texas red (JacksonImmunoResearch Laboratories). PML was detected using 5E10 mAb (for U2OScells) followed by Alexa Fluor 350-conjugated goat anti-mouse Ab(Molecular Probes) or rabbit polyclonal anti-PML Ab (for NIH 3T3 cells)followed Alexa Flour 350-conjugated anti-rabbit Ab (Molecular Probes).eIF4E was detected using FITC-conjugated mouse monoclonal anti-eIF4E Ab(BD Transduction Laboratories). Cells were mounted in Vectashieldsupplemented with DAPI (Vector Laboratories). Images were obtained usingTCS-SP software and displayed using Adobe Photoshop CS 8.0.

Results

eIF4E Physically Associates with Cyclin D1 mRNAs in the Nucleus

To understand the underlying basis for the specificity of eIF4E'seffects on promotion of mRNA export, we examined the novel possibilitythat eIF4E physically associated only with specific mRNAs in thenucleus. In this way, eIF4E-dependent promotion of export of cyclin D1mRNAs could arise through a specific physical interaction of this mRNAwith eIF4E in the nucleus. First, we examined whether eIF4Eimmunoprecipitates with cyclin D1 or housekeeping genes like GAPDH andactin mRNAs in total cell lysates and subsequently in nuclear andcytoplasmic fractions in a variety of cell lines including U2OS, NIH3T3,K562, U937, and HEK293T cells. Results were the same across cell linesso only representative results are shown here (FIG. 11). Note that boththe mRNAs, and the eIF4E examined here, are endogenous. RNAs weredetected for each experiment independently using multiple PCR strategiesincluding quantitative RT-PCR and semi-quantitative PCR. Consistentresults were always obtained using these different methodologies.

Immunoprecipitation studies indicated that in total cell lysates, eIF4Ebound both cyclin D1 and GAPDH mRNAs, as expected because these mRNAsare capped (FIG. 11 a). In the nuclear fraction, eIF4E physicallyassociates with a readily detectable fraction of cyclin D1 mRNA (FIG. 11b). Yet, no detectable association between eIF4E and GAPDH mRNA or actinmRNA is observed in the nuclear fraction in contrast to total celllysates or the cytoplasmic fractions (FIG. 11 a and not depicted). Theseresults are confirmed by our semi-quantitative and independently RT-PCRanalysis (FIGS. 11 b, d, and e). Also, eIF4E associates only withprocessed cyclin D1 mRNAs in the nucleus, as observed using specificprimers and RT-PCR (not depicted). Importantly, the ability of eIF4E toassociate with GAPDH or cyclin D1 mRNAs was monitored using materialfrom the same eIF4E immunoprecipitations. Thus, differences in bindingaffinity between GAPDH and cyclin D1 are not a result of differences inimmunoprecipitation efficiency or in the quality of the fractionationbetween experiments. Controls for the quality of theseimmunoprecipitations and fractionations are given below and discussed inthe Materials and methods.

The above findings suggested that eIF4E-mRNA recognition in the nucleuscould be substantially different to that in the cytoplasm. Inparticular, it was critical to establish the importance of cap bindingfor eIF4E-mRNA recognition in the nucleus. Thus, we examined whichfeatures of eIF4E were required for interaction with cyclin D1 mRNA inthe nuclear fraction using the GST pull-down approach referred to asspecific nucleic acids associated with protein (SNAAP; Trifillis et al.,1999). Here nuclear lysates were incubated with glutathionesepharose-bound wild-type or mutant forms of eIF4EGST or GST (FIG. 11c). Consistent with the immunoprecipitation findings, wild-type eIF4Eassociates with cyclin D1 but not GAPDH mRNAs. No association isobserved with GST (FIG. 11 c) or an unrelated mRNA-binding proteinalpha-CP 1-GST (not depicted) for either mRNA. The W56A eIF4E mutant,which does not bind the cap, does not bind cyclin D1 indicating thateIF4E still requires its cap-binding activity to associate with mRNAs inthe nuclear fraction (FIG. 11 c). We extended these studies to testwhether the dorsal surface mutant, W73A, can still associate with cyclinD1 mRNA in the nucleus, because this mutant readily enhances transportof cyclin D1 when expressed (Topisirovic et al., 2002, 2003a).Importantly, W73A mutant is deficient in translation but not transport.This mutation does not detectably reduce binding to cyclin D1 mRNA ascompared with wild type (FIG. 11 c). Note that previous biophysicalstudies indicate that W56A and W73A mutants have structuresindistinguishable from wild-type eIF4E (Kentsis et al., 2001). Thus,there appears to be a correlation between the ability of eIF4E tophysically associate with cyclin D1 mRNA in the nucleus and the abilityof eIF4E to enhance transport of these mRNAs.

We extended these findings to further demonstrate the requirement forthe m7G cap for association of mRNA with eIF4E in the nucleus (FIGS. 11d and e). We monitored the ability of m7G cap analogue (m7GpppG) tocompete for mRNA binding using semi-quantitative PCR and independently,quantitative RT-PCR methods. Consistent with the above results using theW56A mutant, the cap analogue successfully disrupts the association ofcyclin D1 mRNAs with eIF4E. In contrast, GpppG, which does not bindeIF4E, does not disrupt its association with cyclin D1. In either caseeIF4E does not associate with GAPDH mRNA. Together, these findingsindicate that eIF4E requires the m7G cap in order to associate withspecific mRNAs in the nucleus. Note that treatment with m7GpppG or GpppGdid not alter the amount of eIF4E immunoprecipitated by eIF4E antibodyin these reactions (unpublished data).

Interestingly, when the cytoplasmic fractions of cells were incubatedwith eIF4E-GST, all mRNAs bound, similar to the results we observed forimmunoprecipitation experiments using the total lysates or cytoplasmicfraction (unpublished data). It is of interest that even when nuclearlysates are incubated with recombinant eIF4E in the SNAAP assay, we donot observe an association with GAPDH mRNA (FIG. 11 c). This raises thepossibility that eIF4E-mRNA recognition is restricted in nuclear lysatesby other regulatory elements that are not present in the cytoplasm,where cap binding is sufficient to mediate these interactions. As apositive control, we extended our experiments to determine whether bothGAPDH and cyclin D1 mRNA bound to the other nuclear cap-bindingproteins, CBP 80 and CBP 20 which together form the cap-binding complex(CBC). In general, CBC associates with all transcriptscotranscriptionally (Visa et al., 1996). Immunoprecipitations wereperformed using an antibody to CBP 80. Results were monitored bysemiquantitative and independently by RT-PCR methods. Parallelexperiments were performed with eIF4E antibodies using the same nuclearfractions. As expected, CBC associates with both cyclin D1 and GAPDHtranscripts, whereas eIF4E associates only with cyclin D1 mRNA (FIG. 11f). We further determined whether the CBC associates with eIF4E. Usingimmunoprecipitation (FIG. 11 g) and separately immunofluorescence (notdepicted), we observed no association between the CBC and eIF4E. Thesefindings are consistent with previous reports showing nocoimmunoprecipitation between CBC and eIF4E (Ishigaki et al., 2001;Lejeune et al., 2002). However, we cannot rule out the possibility of atransient interaction between the CBC and eIF4E that we cannot detect bythese methods. Together, these data suggest that eIF4E-cyclin D1 mRNAand CBC-cyclin D1 mRNA complexes are distinct complexes in the nucleus.

We cannot rule out the possibility that, in the nucleus, eIF4E binds alow level of GAPDH mRNA, which is beyond the detection limits of ourRT-PCR methods. Even if this is the case, we readily detect anenrichment of up to 1,000-fold for cyclin D1 relative to GAPDH mRNAsdespite the relative differences in abundance, with GAPDH being the muchmore abundant mRNA in both fractions (FIG. 11 c and see FIG. 13 b).Thus, using two independent methods, immunoprecipitation and SNAAP, wedemonstrate that eIF4E physically associates with specific mRNAs in thenuclear fraction. Furthermore, eIF4E requires its cap-binding activityfor this association but not W73 on the dorsal surface. Recent findingsby another laboratory suggest that eIF4E associates with all mRNAs inthe nuclear fraction (Lejeune et al., 2002), whereas the data we presenthere clearly indicate that eIF4E binds cyclin D1 but not GAPDH or actinmRNAs in the nuclear fraction. The most likely reason for thisdiscrepancy is differences in experimental approach. One majordifference is that we monitor association of eIF4E with endogenous, notoverexpressed, mRNAs (FIG. 11). Overexpression could lead to theformation of RNPs that are different from endogenous RNPs. Thus, weinitiated our studies with endogenous eIF4E as well as endogenous mRNAs.Detection of bound mRNAs in immunoprecipitated fractions is alsocritical for optimal interpretation of these experiments. We confirmedour results with quantitative RT-PCR methods in order to ensure thatbackground binding of mRNAs was not mistaken for real binding.Furthermore, we obtain the same results using different eIF4E antibodiesor reconstituting the complexes with eIF4E-GST. Clearly, our specificitycorrelates well with previous observations by our group and theSonenberg group that eIF4E overexpression up-regulates cyclin D1 but notGAPDH or actin mRNA transport and correspondingly up-regulates cyclin D1but not GAPDH and actin protein levels.

Cyclin D1 mRNAs are Localized to a Subset of eIF4E Nuclear Bodies

Because eIF4E specifically associates with cyclin D1 mRNA in thenucleus, we examined whether cyclin D1 mRNA specifically associates witheIF4E nuclear bodies. In this way, eIF4E nuclear bodies could be sitesof assembly of specific RNPs or functional storage sites. Studies wereperformed in U2OS and NIH3T3 cells. The localization of cyclin D1 orGAPDH mRNAs was determined using in situ hybridization and thelocalization of eIF4E and another component of the nuclear body, PML,through immunofluorescence. The results were monitored using confocalmicroscopy. Similar results are observed in both U2OS and NIH3T3 cells(FIGS. 12 a and b). These studies reveal that cyclin D1 mRNAs (red) arefound throughout the cytoplasm and nucleoplasm but are additionallyenriched in bodies in the nucleus. These local sites of enrichmentcolocalize with a subset of eIF4E nuclear bodies (green). Sites ofcolocalization of eIF4E nuclear bodies and cyclin D1 mRNAs are shown inyellow with two of several such sites marked with arrows (FIG. 12). Notethat the objective for all experiments in FIG. 12 was 100× with furthermagnifications as follows: twofold for A-C; and 1.5-fold for D. Thecurrent resolution of these studies does not enable us to distinguishwhether cyclin D1 mRNAs are found on the surface or within the eIF4Ebodies. Consistent with previous studies (Lai and Borden, 2000; Cohen etal., 2001), there are two populations of eIF4E nuclear bodies, those,which contain PML, and those, which do not. The majority of eIF4E(green) and PML (blue) colocalize to the same nuclear bodies (lightblue) and, as observed previously for many cells, there are additionaleIF4E bodies (FIG. 12 a, green; Lai and Borden, 2000; Cohen et al.,2001).

Importantly, mRNAs were never observed to colocalize with PML nuclearbodies consistent with previous studies showing RNA did not localizewith PML nuclear bodies (Boisvert et al., 2000). Thus, cyclin D1 mRNAslocalize to the subset of eIF4E nuclear bodies that do not contain PML.As expected, GADPH mRNAs do not localize with either PML or eIF4Enuclear bodies (FIG. 12 b). These results are consistent with theobservation that nuclear GAPDH mRNAs do not physically associate witheIF4E and do not have their export modulated by eIF4E overexpression(Topisirovic et al., 2002, 2003a). As a negative control, probes forcyclin D1 in situ hybridization in cyclin D1−/− cells revealed no signalindicating that these probes are specific for cyclin D1 (FIG. 12 c).Furthermore, RNase treatment completely abolishes signals (notdepicted). As expected given the above results, immunoprecipitationstudies with a PML antibody reveal no association with either cyclin D1or GAPDH mRNAs. These data are consistent with our previous findingsthat PML reduces the affinity of eIF4E for the m7G cap by >100-fold(Kentsis et al., 2001), thus disabling RNA binding. Because eIF4Erequires its cap-binding activity for interaction with cyclin D1 (FIGS.11 d and e), it is consistent that cyclin D1 mRNAs are not found at PMLcontaining eIF4E nuclear bodies.

In summary, cyclin D1 mRNAs localize to a subset of eIF4E nuclearbodies. Localization of mRNAs to the bodies is specific and is likely tobe functionally important for their subsequent transport to thecytoplasm. In this way, eIF4E nuclear bodies may be assembly sites forspecific eIF4E-RNPs, which enable promotion of export to the cytoplasm.Furthermore, it appears that, in the nucleus, there must be featuresparticular to the bound mRNAs that impart the observed specificity ofeIF4E.

Physical Association of eIF4E with mRNAs is Correlated with EnhancedmRNA Transport

Above, we demonstrate that both wild-type eIF4E and the W73A mutantphysically associate with cyclin D1 mRNA in the nuclear fraction butthat the W56A mutant, which is deficient in cap binding, does not (FIG.11 c). To determine whether there is a correlation between binding andmRNA transport, we assessed the ability of these mutants to promotetransport of cyclin D1 mRNA. Stably transfected NIH3T3 cells expressingmutant or wild-type proteins were fractionated and mRNAs monitored byNorthern analysis (FIG. 13 a and Table I) as described previously(Topisirovic et al., 2002). U6snRNA and tRNALys serve as fractionationcontrols. Note that GAPDH is not altered in any case, as expected.Furthermore, the mutant proteins are expressed to similar levels (FIG.13 c) and total levels of cyclin D1 mRNA are not altered by any of theconstructs (FIG. 13 b). Furthermore, the stability of the cyclin D1transcript is not affected by eIF4E (FIG. 13 d and Table II).

Importantly, eIF4E and the W73A mutant promote cyclin D1 mRNA transportwhere more cyclin D1 transcripts are clearly visible in the cytoplasmicfractions versus vector controls. Importantly, the W56A mutant does notalter the subcellular distribution of cyclin D1 mRNA transcripts (FIG.13 a and Table I). One of the consequences of eIF4E-dependent mRNAtransport is increased protein levels due to higher concentrations ofthese mRNAs in the cytoplasm and thus increased availability of thesemRNAs to the translational machinery. Consistent with the abovefractionation studies, cyclin D1 protein levels are elevated inwild-type and W73A mutant experiments but there is no increase when theW56A mutant is overexpressed. Thus, the physical association of cyclinD1 mRNA with the nuclear fraction of eIF4E is strongly correlated withthe enhanced transport of cyclin D1 mRNA from the nucleus to thecytoplasm.

PML overexpression leads to the nuclear retention of cyclin D1 but notGAPDH mRNAs (FIG. 13 a), as well as reduced cyclin D1 but not GAPDH oractin protein levels (FIG. 13 c). This is consistent with the resultsfrom immunoprecipitation and in situ studies, where PML inhibitsformation of eIF4E-cyclin D1 mRNA complexes (FIG. 11 b and FIG. 12 a).Once again it links the ability of eIF4E to physically interact withRNAs to the ability to promote mRNA transport. Previous studiesdemonstrated that eIF4E could enter the nucleus by interaction with the4E transporter protein (4ET; Dostie et al., 2000). Here, mutation of thedorsal surface (W73A) impaired association with the 4ET and thusimpaired nuclear entry (Dostie et al., 2000). Thus, we performedexperiments to ensure that the W73A mutant still entered the nucleus andformed nuclear bodies (FIG. 13 e). Using confocal microscopy, weexamined the subcellular distribution of overexpressed eIF4E or the W73Amutant using the Xpress epitope tag and additionally an antibody toeIF4E, which recognizes both endogenous and overexpressed protein.

It is clear from the confocal micrographs that the W73A mutant isreadily detectable in the nucleus and associates with endogenous eIF4Enuclear bodies (FIG. 13 e). Thus, it appears that when the W73A mutantis overexpressed it uses an alternate route or can overcome the weakerbinding to 4ET, gets transported into the nucleus and associate withnuclear bodies (FIG. 13 e). Similar studies with the W56A mutantindicated no alteration in subcellular distribution as compared withwild type (not depicted). In addition, wild-type and mutant forms ofeIF4E are expressed to similar levels (FIG. 13 c). eIF4E levels areexpressed to similar levels (FIG. 13 c). Note that the objective was100× for these micrographs with a further 1.5-fold magnification.

TABLE I Relative Nc/Cyt ratio of cyclin D1 mRNA in cells transfected asindicated (densitometry analysis of Northern blot experiments)a Vector1.110 +/− 0.490 WT4E  0.171 +/− 0.0828 W56A4E 1.194 +/− 0.365 W73A4E0.216 +/− 0.102 PML 4.552 +/− 0.632 a- Cyclin D1 mRNA levels werenormalized to GAPDH mRNA; +/− value represents SD from three independentexperiments.

TABLE II Relative cyclin D1 mRNA level after actinomycin D treatment ofcells transfected with eIF4EWT or vector control (measured by RT-PCR) -a Time 4EWT Vector 0    1 +/− 0.3422    1 +/− 0.5820 2 h 0.1654 +/−0.04   0.1314 +/− 0.04600 4 h 0.0718 +/− 0.0120 0.0567 +/− 0.0108 6 h0.0199 +/− 0.0028 0.0218 +/− 0.0084 a - Cyclin D1 mRNA levels werenormalized to GAPDH mRNA; +/− value represents SD from three independentexperiments.Identification of an RNA Structural Element that Mediates eIF4ESensitivity in the Nuclear Compartment

To determine if the association of mRNAs with eIF4E in the nucleus andeIF4E-dependent mRNA transport are mediated through some specific mRNAsequence, we analyzed 3′ and 5′ UTRs from our model mRNA cyclin D1. Aseries of chimeric constructs were made fusing the coding region of LacZto the 5′ or 3′ UTRs of cyclin D1 (FIG. 14 a). We assessed whether thesesequences were necessary and sufficient to enable chimeric mRNAs toassociate with endogenous eIF4E in the nucleus and subsequently havetheir export modulated. Experiments were performed in NIH3T3 and HEK293Tcells, which gave identical results. Note that HEK293T cells formnuclear bodies similar in size and morphology to those observed forNIH3T3 cells (FIG. 12 d). Initial semi-quantitative PCR results wereconfirmed by quantitative RT-PCR methods using the standard curvesmethod (FIG. 14 b). We monitored the ability of the nuclear fraction ofeIF4E to associate with these mRNAs using immunoprecipitation inconjunction with PCR (FIG. 14 b). Importantly, eIF4E does notimmunoprecipitate with LacZ mRNA, does not immunoprecipitate with LacZ-cyclin D1 5′ UTR chimeric mRNA, but does associate with chimeric LacZmRNA that contains the entire 3′UTR of cyclin D1. We made additionalchimeric LacZ constructs with two different parts of the 3′ UTR using anEcoRI site positioned approximately in the center of 3′ UTR of cyclin D1cDNA, and showed that chimeric RNA that contains first part of cyclin D13′ UTR (3′ UTRA) immunoprecipitates with nuclear eIF4E whereas thesecond part (3′ UTRB) does not.

Analysis of additional chimeric constructs containing different elementsfrom the first part of cyclin D1 3′ UTR revealed that the 100-bpsequence from the 3′ UTR of cyclin D1 (located 2,471-2,565 bp in humancyclin D1 cDNA) is necessary and sufficient for association with eIF4E,so we refer to it as an eIF4E-sensitive element (4E-SE). Importantly,this element is the highly conserved between human, mouse, rat, andchicken sequences (FIG. 14 c). In fact, the 4E-SEs between humans andchicken are nearly identical with 94% conservation versus 59% similarityover the rest of the 3′UTR.

The presence of the 4E-SE in mammals and birds suggests that it isevolutionarily conserved. To assess if the interaction of chimeric mRNAswith eIF4E was functional, we examined the effects of eIF4E expressionon their export (FIG. 15). mRNA export was monitored using subcellularfractionation in conjunction with semiquantitative RT PCR (FIG. 15 a),northern methods (FIG. 15 b through 15 d) or quantitative RT-PCR (TableIII). eIF4E does not modulate the transport of LacZ or LacZ chimerasthat do not contain the 4E-SE (FIG. 15 and Table III), which isconsistent with the observation that eIF4E does not bind these mRNAs(FIG. 14 b). Note that total mRNA levels determined from the sametransfected cells indicated that LacZ mRNAs levels were not modulated(FIG. 16 b) nor were their stability (FIG. 16 c). Thus, there is astrong correlation with the ability of eIF4E to associate (directly orindirectly) with the 3′ UTRs of these mRNAs and promote their transport.Increased export of LacZ mRNA, and thus the higher levels of cytoplasmicmRNAs when the 4E-SE is present, is correlated with higher levels ofLacZ protein (FIG. 16 a). Consistent with our earlier observations,overexpression of the W56A mutant does not alter transport of eitherLacZ or LacZ-4E-SE as compared with wild-type eIF4E nor did the W56Amutant alter protein production of either LacZ construct (FIG. 15 d andFIG. 16 a; Table III). Thus, the mRNAs retain their cap dependence.Furthermore, all of the chimeric constructs had similar levels of totalmRNA indicating that differences observed at the protein level wereposttranscriptional and that differences in association with eIF4E andtransport were not due to differences in expression of the constructs(FIG. 16 b). Importantly, LacZ-4E-SE transport is negatively regulatedby PML (FIG. 16 a), as we observed for endogenous cyclin D1 mRNA (FIG.13 a through c). Together, these results indicate that both the 4E-SEand the m7G cap are required for eIF4E to enhance transport of thesemRNAs.

TABLE III Relative ratio of cytoplasmic versus nuclear LacZ mRNA ofcells transfected as indicated (measured by RT-PCR)a Construct RelativeC:N LacZ mRNA ratio 4EWT +/− LacZ    1 +/− 0.082 4EWT +/− 3′ UTR4462.496 +/− 38.114  4EWT +/− 3′ UTR Full 373.934 +/− 30.195  4EWT +/− 3′UTR 2/3 0.823 +/− 0.069 4EWT +/− 3′ UTR B 1.187 +/− 0.119 W56A +/− lacZ1.159 +/− 0.124 W56A +/− 3′ UTR 4 1.918 +/− 0.286 a- LacZ mRNA levelswere normalized to GAPDH mRNA +/− value represents SD from threeindependent experiments.The 4E-SE Contributes to eIF4E Mediated Oncogenic Transformation

We extended these studies to establish whether the 4E-SE contributed tothe physiological activities of eIF4E and thereby to assess thefunctional significance of this RNA element (FIG. 17). Our previousstudies correlated eIF4E-dependent promotion of cyclin D1 mRNA exportwith the transformation activities of eIF4E so we examined thecontribution of the 4E-SE to this activity. Transformation activity wasassessed by monitoring the number of foci formed upon eIF4Eoverexpression in a cyclin D1−/− fibroblast cell line. Note that thedistribution of eIF4E nuclear bodies is not altered in cyclin D1−/− ascompared with other cell types (FIG. 12 c). First, we determined thateIF4E transformed cyclin D1−/− cells relative to vector controls.Reintroduction of cyclin D1 constructs containing the full-length 3′UTR(cycFull) led to substantially more foci than cells transfected witheIF4E alone (FIG. 17 a). However, eIF4E's transformation activity wasnot augmented by introduction of cyclin D1 with no 3′UTR (cycTrunc)being the same as eIF4E overexpressing cells alone. Importantly,introduction of eIF4E and cyclin D1, with only the 100 nt 4E-SE(cyc4E-SE), transformed cells as well as constructs containing thefull-length 3′UTR. Thus, in the context of cyclin D1−/− cells, thetransformation activity of eIF4E is only increased by reintroduction ofcyclin D1 when the 4E-SE is present. Consistently, only those cellstransfected with cyclin D1-3′UTR (cycFull) or cyclin D1-4E-SE (cyc4E-SE)showed increased cyclin D1 protein levels in contrast to vector controlsor cells transfected with cyclin D1 with truncated 3′UTR (cyc-Trunc;FIG. 17 b).

Thus, the presence of the 4E-SE is tightly tied to eIF4E's ability toexport cyclin D1 and subsequently to efficiently transform cells. Theseeffects can be extended to endogenous eIF4E. Cells expressing cycFull orcyc4E-SE, even in the absence of overexpressed eIF4E, produce morecyclin D1 protein than those cells expressing the truncated version ofcyclin D1 (FIG. 17 b). We confirm this is occurring at the mRNAtransport level by fractionation and RT-PCR methods (FIG. 17 c). Wedemonstrate that the ratio of nuclear to cytoplasmic cyclin D1 mRNA is˜250 times greater in those cyclin D1−/− cells expressing the cycTruncconstruct than those expressing the cycFull or cyc4E-SE constructs.Thus, the cycTrunc is not as efficiently transported to the cytoplasm ascycFull and cyc4E-SE constructs are. Importantly, the distribution ofGAPDH was not altered by any of these constructs (unpublished data).Northern analysis confirmed these findings and indicated thatfractionations were clean (unpublished data). Thus, the presence of the4E-SE allows more efficient export of cyclin D1 mRNA using eitherendogenous or exogenous eIF4E.

Discussion

These studies reveal that eIF4E associates with and regulates nuclearmRNAs in a fundamentally different manner than cytoplasmic mRNAs. Unlikethe cytoplasmic fraction of eIF4E where cap binding is sufficient forits functional interaction with mRNAs, in the nucleus eIF4E appears toassociate with regulatory factors that restrict its association withmRNA lacking 4E-SEs. Because eIF4E binds the m7G cap, we hypothesizethat other factors directly bind the 4E-SE in the 3′UTR and throughphysical association with eIF4E increase its affinity for this subset ofmRNAs (FIG. 18). An mRNA looping model is another possibility, whereeIF4E cap binding is stabilized by direct contact with the 4E-SE,through an unknown mechanism (FIG. 18). It seems likely that not onlycyclin D1 but also many other mRNAs could be regulated in this way(unpublished data), especially given that ODC also has its transportregulated in this manner (Rousseau et al., 1996). Our studies and recentreports indicate that eIF4E does not associate with the CBC nor does itassociate with unspliced mRNA (Ishigaki et al., 2001; Lejeune et al.,2002). These studies suggest that the transfer of capped cyclin D1 mRNAtranscripts from the CBC to eIF4E happens after splicing and beforecyclin D1 mRNA gets exported from the nucleus. Because eIF4E and CBC donot coimmunoprecipitate or colocalize, this interaction is likelytransient one. We cannot rule out the possibility of a completely novelmechanism by which the cap of cyclin D1 mRNA is protected by someunknown means between leaving the CBC RNP and associating with eIF4E.This is an area of future investigation.

mRNAs that get exported in an eIF4E-dependent fashion may undergo somealternative, eIF4E-dependent type of mRNA quality surveillance. Previousstudies suggested that the nuclear fraction of eIF4E might be involvedin low level nuclear translation as part of mRNA quality surveillance(Iborra et al., 2001). However, our studies with the W73A mutantindicate that nuclear translation is not required for the observedtransport function because this mutant is active in transport but nottranslation, because it cannot bind eIF4G (Sonenberg and Gingras, 1998;Gingras et al., 1999). Specialized pathways for transport ofgrowth-promoting mRNAs such is cyclin D1, and control of this process byfactors such as PML, may have evolved in order to coordinate geneexpression with cellular proliferation. eIF4E nuclear bodies must beintact in order to act in mRNA export because their disruption iscorrelated with a loss of export activity (Topisirovic et al., 2003a;Kentsis et al., 2004). Our data suggest that assembly of eIF4E transportRNPs happens in or around eIF4E bodies. The colocalization of cyclin D1mRNAs with PML-negative eIF4E nuclear bodies suggests that these sitesare areas for assembly of specific subtypes of RNPs which permit moreefficient export of this restricted subset of mRNAs to the cytoplasm. Inthis way, expression of these targeted mRNAs could be modulated quitequickly. It seems likely that nuclear eIF4E RNPs involved in promotionof mRNA export are different from those functioning in translation,because the W73A mutant is still active in transport (Cohen et al.,2001; Topisirovic et al., 2003a). Consistently, eIF4E does not appear tobind eIF4G in the nucleus (McKendrick et al., 2001) but eIF4G is anintegral part of the eIF4E RNP in the cytoplasm (Sonenberg and Gingras,1998). Clearly these results suggest major differences infunctionalities of the corresponding nuclear and cytoplasmic eIF4E RNPs.eIF4E-dependent promotion of mRNA export could provide an immediateresponse system by which the cell responds to stress and/or growthconditions before transcriptional reprogramming.

We speculate that this process is not limited just to cyclin D1 mRNA butthat other mRNAs involved in growth regulation could be regulated thisway, including ODC (Rousseau et al., 1996) and many others (unpublisheddata). The ability of eIF4E to promote the export of growth promotingmRNAs such as cyclin D1 allows it to turn on a cellular growth promotingprogram thereby positioning eIF4E as a critical node in the growthregulatory network. eIF4E regulating proteins, such as PML (this paper)and nuclear homeodomain proteins such as PRH, which directly bind eIF4E(Topisirovic et al., 2003a) are well positioned to act upstream ofeIF4E. Although, this network also includes important regulation oftranslation by the eIF4E-binding proteins (4EBPs; Sonenberg and Gingras,1998), our findings suggest that these transport and translationnetworks may not completely overlap. For instance, cyclin D1 mRNA issensitive to eIF4E at the transport level, but not at the translationlevel (Rousseau et al., 1996). In contrast, ODC mRNA is sensitive toeIF4E at both levels (Rousseau et al., 1996). ODC mRNA, like cyclin D1mRNA, contains a 4E-SE element (unpublished data). PML appears to be acritical negative regulator of this nuclear network, thereby shuttingdown production of a wide variety of growth promoting proteinssimultaneously and thus, inhibiting eIF4E-mediated growth andtransformation. These activities rely on eIF4E RNA recognition throughboth the m7G cap and the 4E-SE. eIF4E promotion of export of specificmRNAs represents an exciting new point of growth regulation in the celland a novel regulatory pathway which when dysregulated could contributeto human cancers.

Example 4 eIF4E is a Central Node of a RNA Regulon that Governs CellularProliferation

Abstract

Eukaryotic translation initiation factor eIF4E is a critical node in anRNA regulon that impacts nearly every stage of cell cycle progression.Specifically, eIF4E coordinately promotes the mRNA export, and in somecases also translation, of several genes involved in cell cycleprogression. A common feature in these mRNAs is a structurally conserved˜50 nucleotide element in the 3′UTR denoted an eIF4E sensitivity element(4E-SE). This element is sufficient for localization of capped mRNAs toeIF4E nuclear bodies, formation of eIF4E specific RNPs in the nucleus,and eIF4E dependent mRNA export. These studies indicate that roles ofeIF4E in translation and mRNA export are distinct, relying on differentsequence elements in mRNA and formation of distinct RNPs. Furthermore,eIF4E dependent mRNA export is independent of on-going RNA or proteinsynthesis. Unlike export of bulk mRNAs, which is NXF1 dependent, eIF4Edependent mRNA export is CRM1 mediated. These data provide a novelperspective into the molecular mechanism of the proliferative andoncogenic properties of eIF4E.

Introduction

RNA regulons have been proposed as a means by which eukaryotic cellscoordinate gene expression. In contrast to prokaryotes where coordinatedregulation of genes is achieved by genomic organization, eukaryotescoordinate the regulation of subsets of mRNAs involved in the samebiological processes at the post-transcriptional level by manipulatingcompositions and activities of discrete subsets of RNPs. It has beenpostulated that related RNA sequences termed “untranslated sequenceelements for regulation” (USER codes), similar to zipcodes for RNAlocalization, are used for specific association with variety ofregulatory proteins involved in different levels of post-transcriptionalregulation. mRNA nuclear export is one level of control that could becoordinated in this way. Initially, mRNA export was thought to be ageneral process by which all mRNAs were transported from the nucleus tothe cytoplasm irregardless of sequence specific features. More recentfindings indicate that mRNA export can be coordinated with other eventsin RNA metabolism, particularly transcription and splicing, and thus,that nuclear history of transcripts can modulate the cytoplasmic fate oftargeted mRNAs. This way, nuclear export can be coordinated throughcompartmentalization via mRNP organization, coupling coordinated exportof functional classes of mRNAs with their functions in biologicalprocesses such as proliferation, differentiation and development.

Studies with eukaryotic translation initiation factor eIF4E provide anexample of a factor that differentially effects expression of a subsetof mRNAs. Even though it associates with all transcripts through thecommon 5′ methyl-7-guanosine (m⁷G) cap structure, many groups showedthat eIF4E overexpression does not lead to global increases in proteinexpression. In the cytoplasm, mRNAs deemed eIF4E sensitive have theirprotein levels modulated by eIF4E more so than other mRNAs. Thissensitivity is attributed to the complexity of the 5′UTRs in thesetranscripts. Up to 68% of eIF4E is found in the nucleus in a broadvariety of species ranging from yeast to humans. Here, eIF4Eoverexpression leads to increased export of cyclin D1 but not GAPDHmRNA. Specific association of eIF4E with cyclin D1 mRNA in the nucleusrequires the m⁷G cap, and a small element in its 3′ UTR referred as aneIF4E sensitivity element (4E-SE).

Overexpression of eIF4E is correlated with oncogenic transformation intissue culture, cancers in animal models, and poor prognosis in a numberof human cancers. Several lines of evidence suggest that the mRNA exportfunction of eIF4E contributes to its oncogenic potential. For instance,cyclin D1 mRNA export is upregulated in specific subtypes of humanleukemia. These specimens contain unusually high levels of eIF4E, thevast majority of which is located in the nucleus. Also, inhibitors ofeIF4E dependent mRNA export, the promyelocytic leukemia protein (PML)and homeoprotein PRH, bind eIF4E in the nucleus, inhibit eIF4E dependentmRNA export and eIF4E mediated oncogenic transformation. Further,mutagenesis studies strongly link the activity of eIF4E in mRNA exportto its ability to oncogenically transform cells.

Although cyclin D1 plays a key role in the cell cycle that links eIF4E'sproliferative properties and its mRNA export function, it is possiblethat eIF4E coordinately alters the expression of some other growthpromoting mRNAs as well, in order to drive its proliferative potential.This study shows that several mRNAs involved in cell cycle progressionare also targets of eIF4E dependent mRNA export, and that the subsets ofmRNAs regulated at the level of eIF4E dependent mRNA export are distinctfrom those that are preferentially translated in the cytoplasm. Weidentified an underlying USER code for export of eIF4E sensitivetranscripts. This code is required for the subnuclear distribution ofthese RNAs, as well as for the formation of relevant eIF4E RNPs.Interestingly, the 4E-SE USER code is a structurally conserved elementrather than a sequence based one. eIF4E dependent mRNA export can bedecoupled from translation. Finally, eIF4E dependent mRNA export occursvia an alternative mRNA export pathway than bulk mRNA. These resultsprovide the basis for a novel paradigm for eIF4E-mediated tumorigenesis.

Materials and Methods

Reagents and constructs. Chimeric constructs in pcDNA3. LacZ vector(Invitrogen) were positioned 3′ of the coding region of LacZ. Cyclin D1minimal 4E-SE (c4E-SE) was amplified using primers containing EcoRI orXbaI restriction sites at the 5′ ends, and the LacZ3′UTR construct as atemplate (Culjkovic et al, 2005). The same approach was used for thecloning of Pim-1 constructs, where pRBK-Pim-1 (kind gift from NancyMagnuson (Hoover et al., 1997) was used as a template. Primer sequencesare available in Supplemental Table 1. For the TetON system, chimericLacZ constructs were cloned into pTREMyc vector (Clontech) using EcoRIand XbaI. pcDNA2Flag-eIF4E, pMV, pMV-eIF4E wild type or mutants,pLINKSV40-PML, MSCV, MSCV-eIF4E WT or mutants and bacterial expressionconstructs were previously described (Cohen et al., 2001; Culjkovic etal., 2005; Topisirovic and Borden, 2005; Topisirovic et al., 2003b).Reagents used were all analytical grade from Sigma, unless mentionedotherwise.

Antibodies. Antibodies for immunoblotting: mAb anti-PML (5E10 (Stuurmanet al., 1992)), mAb anti-eIF4E (BD PharMingen), mAb anti-cyclinDI (BDPharMingen), mAb anti-Xpress (Invitrogen), rabbit pAb anti-cyclin E1(M20, Santa Cruz Biotechnology), mAb anti-GAPDH (MAB374, Chemicon), mAbanti-c-Myc (9E10 Santa Cruz Biotechnology), rabbit pAb anti-cyclin A(C-19, Santa Cruz), rabbit pAb anti-nibrin (Cell Signaling), mAbanti-Pim-1 (19F7 Santa Cruz) and mAb cyclin-B1 (GNS1 Santa Cruz).

Cell culture and Transfection. eIF4E and PML stably transfected NIH3T3and U937 cells were as described (Topisirovic et al., 2002; Topisirovicet al., 2003a). U937 cells were used to analyze endogenous Pim1, whichis not expressed in NIH3T3 cells. LacZ/LacZ-4E-SE with or without2Flag-eIF4E as well as TetON LacZ system were stably transfected in U2OScells. For NXF1 depletion, U2OS cells were transfected withLipofectamine 2000 and 10 nM siRNA duplex HSC.RNAI.N006362.1.3 (IDT)according to the manufacturer's instruction. Cells were analyzed 72 hafter transfection. Actinomycin D, cycloheximide and leptomycin B wereall cell culture grade (Sigma).

Immunopurification of eIF4E and RT-PCR. Immunopurification was aspreviously published (Culjkovic et al., 2005). Real Time PCR analyseswere performed using Sybr Green PCR Master mix (ABI) in Mx3000PTMthermal cycler (Stratagene), and data analyzed with MxPro software(Stratagene). All conditions were described previously (Culjkovic etal., 2005). All calculations were done using the relative standard curvemethod described in Applied Biosystems User Bulletin #2.

Differential display of immunopurified RNA was performed using RNAimage™kit (GeneHunter Corporation) according to the manufacturer'sinstructions.

SNAAP protocol used for differential display was performed as described(Trifillis et al., 1999).

Western blots were performed as described (Topisirovic et al., 2002;Topisirovic et al., 2003a).

Cellular Fractionation and Northern Analysis. Fractionation and RNAisolation were as described (Lai and Borden, 2000; Topisirovic et al.,2002). Probes for U6 and tRNAlys for Northern blot analysis werepreviously described (Topisirovic et al., 2002).

Immunofluorescence and Laser Scanning Confocal Microscopy. Experimentswere as described (Cohen et al., 2001; Topisirovic et al., 2002).Fluorescence was observed using 100× optical magnification and 3 or 4×digital zoom, as indicated, on LSM 510 Meta (Carl Zeiss Jena) invertedlaser scanning confocal microscope exciting at 488, 543 or 405 nm (atRT). All channels were detected separately, and no cross talk betweenthe channels was observed. The confocal micrographs represent a singleoptical section through the plane of the cell.

In situ hybridization was as previously described (Culjkovic et al.,2005) using nick translated Biotin-11-dUTP-labeled probes (NickTranslation kit, Roche). Probes were detected using Cy3 IgG Fractionmouse mAb Anti-Biotin (1:100; Jackson ImmunoResearch Laboratories).

EMSA analyses were performed as published (Wein et al., 2003) with thefollowing modifications: 20-50 μg nuclear lysate were incubated with32P-3′ end labeled LacZ, LacZ-c4E-SE or LacZ-p4E-SE transcript (˜50000cpm) in 25 μl NET-2 buffer supplemented with 5 mg yeast tRNA (Sigma) and3 mM MgCl₂ for 30 min at room temperature with an additional 15 minutesafter addition of 2 mg/ml heparin. For competition studies thenonlabeled competitor RNAs were pre-incubated for 10 minutes withnuclear lysates before labeled RNAs were added. For supershiftexperiments nuclear lysates were pre-incubated with mAb anti-eIF4E (BDPharMingen) for 15 minutes prior to addition of labeled RNAs.Immunodepleated lysates were from IPs with rabbit pAb anti-eIF4E(Abcam). All mRNAs were in vitro transcribed using mMessage mMachine™ T7kit (Ambion) and 3′ end labeled using [³²P]pCp and T4 RNA Ligase(Amersham). Samples were separated by electrophoresis on 5% native(19:1) polyacrylamide gels for 2 h at 250V using 1× Tris-Borate-EDTAbuffer.

UV crosslinking. 50 μg of nuclear lysates were incubated withradiolabeled probes (1-2×10⁵ cpm) using the same conditions as for theEMSA. After incubation with heparin, samples were placed on ice and UVirradiated for 15 minutes in a Stratalinker UV 1800 (Stratagene).Crosslinked RNA:protein complexes were treated with 10U RNase A and 10URNase T1 for 15 minutes at 37° C. The reactions were stopped by theaddition of 30 μl 2×SDS sample buffer and heating 10 min 95° C. Sampleswere loaded on 10 or 12% SDS polyacrylamide gels and separated at 50Vfor 16 h at RT.

RNase mapping analyses were performed as described (Clever et al., 1995)and according to the manufacturer's instructions (Ambion). Briefly,approximately 0.5-1×105 cpm 32P-5′-end labeled c4ESE or p4ESE RNA oligoprobes (IDT) were mixed with 3 μg yeast tRNA and incubated with 1, 0.1or 0.01U RNase VI (Ambion) for 15 minutes at RT; 1, 0.1 or 0.01U RNase A(Ambion) for 5 min at RT; 1, 0.1 or 0.01U RNase T1 (Sigma) for 15 minRT; 1, 0.1 or 0.01U RNase T2 (Invitrogen) for 5 min RT, or alkalinebuffer for 1, 2 or 5 min at 95° C. (alkaline hydrolysis). Reactions werestopped by EtOH/NaAc precipitation. Samples were resolved on 6%polyacrylamide-8M urea gels in 1×Tris/Borate/EDTA buffer.

Results and Discussion

eIF4E Alters the mRNA Transport of a Wide Variety of Transcripts

eIF4E dependent mRNA export is potentially a broadly based mechanism bywhich eIF4E controls gene expression and thereby modulates growth andproliferation. We sought to determine if mRNAs other than cyclin D1might be regulated in an eIF4E dependent manner. Using nuclear lysates,we isolated mRNAs associated with endogenous eIF4E viaimmunoprecipitation or with recombinant eIF4E using a GST pulldown basedmethod (the SNAAP method of (Trifillis et al., 1999)) and identifiedthem by differential display. Given that many of the identified genesare involved in cell cycle progression, eIF4E immunoprecipitatedfractions were also tested for other genes known to be involved in thisprocesses, as well as for known growth inhibitory mRNAs (Table IV). Alltarget identification was confirmed by eIF4E immunoprecipitation andquantitative or semi-quantitative RT-PCR analysis (FIG. 18 a).Importantly, the list provided in Table IV is not intended to be totallyinclusive but rather to represent a sampling of the target mRNApopulation, since results from differential display data suggest thathundreds of mRNAs are likely regulated in this manner; here weidentified only a subset of these (data not shown).

Many of the mRNAs that physically associate with the nuclear fraction ofeIF4E code for gene products that act in cell cycle progression andsurvival, consistent with the physiological functions associated witheIF4E (see Table IV). Importantly, eIF4E does not bind all mRNAs tested(Table IV). For instance, eIF4E does not associate with the mRNAscorresponding to negative regulators of growth such as PML or p53, orhousekeeping genes such as GAPDH, β-actin or α-tubulin. Also, thisspecificity is not a simple reflection of the sensitivity of mRNAs forregulation at the translational level, as mRNAs sensitive only at thetranslation level (such as VEGF (Clemens and Bommer, 1999)) are notassociated with the nuclear fraction of eIF4E (Table IV). It isimportant to note that mRNAs that were not found in the eIF4Eimmunoprecipitated fractions were readily detected in our nuclearlysates (Table IV). Note that that the estimated efficiency of IP withanti-eIF4E mAb is up to 80%.

Since eIF4E associates with the m⁷G cap of mRNAs, we examined whetherthis was required for the association of eIF4E with mRNAs in the nuclearfraction (FIG. 18 a). eIF4E was immunoprecipitated from the nuclearfraction and mRNAs treated with excess m⁷GpppG or an analogue that doesnot bind eIF4E, GpppG. All mRNAs tested associate with eIF4E in a capdependent manner i.e., m⁷GpppG competes for binding whereas GpppG doesnot. These data indicate that the association of eIF4E with mRNAs in thenucleus is m⁷G cap dependent.

Physical Association of eIF4E with mRNAs is Correlated with EnhancedmRNA Export

To test whether there is a correlation between the ability of eIF4E toassociate with mRNAs in the nuclear fraction and the ability of eIF4Eoverexpression to enhance eIF4E dependent mRNA export, the subcellulardistribution of identified mRNAs as a function of eIF4E overexpressionwas analyzed (Table V). U937 and NIH3T3 cells overexpressing eIF4E orappropriate mutants were fractionated and mRNAs levels monitored by realtime PCR or Northern analysis. eIF4E overexpression increases the amountof eIF4E sensitive mRNAs in the cytoplasmic fraction versus vectorcontrols (Table V). Conversely, transcripts that did not associate witheIF4E in the nuclear fraction did not have their export altered by eIF4Eoverexpression (Table V). As expected, the subcellular distribution ofβ-actin, GAPDH, U6snRNA and tRNA_(Lys) were unaffected (Table V). Thereis no alteration in total mRNA levels (data not shown). Consistently,when eIF4E could not bind these mRNAs because of a mutation in itscap-binding site (W56A), the subcellular distribution of these mRNAs isnot altered (Table V). Further, the dorsal surface mutant W73A whichdoes not act in translation but promotes cyclin D1 mRNA export(Sonenberg and Gingras, 1998; Topisirovic et al., 2002), also promotesexport of other eIF4E sensitive mRNAs (Table V). Thus, it is likely thatall sensitive mRNAs will require the m⁷G cap binding activity of eIF4Ebut not w73 on the dorsal surface for their interaction with eIF4E inthe nucleus. Importantly, circular dichroism studies indicate that bothW73A and W56A mutants have structures indistinguishable from wild typeeIF4E (Kentsis et al., 2001).

One of the consequences of eIF4E dependent promotion of mRNA export isincreased availability of these mRNAs to the translation machinery,leading to increased protein levels. Thus we examined if protein levelsfor a subset of identified genes are elevated by eIF4E. Consistent withenhanced mRNA export, overexpression of wild type eIF4E or the W73Amutant leads to increased protein levels of a subset of genes examined(FIG. 18 b), while there is no increase in protein levels when the capbinding mutant (W56A) is overexpressed. Importantly, wild type eIF4E andthe W73A and W56A mutants were expressed to similar levels for allexperiments (FIG. 18 b).

In order to determine if these mRNAs are regulated through the samemechanism, it was important to examine the effect of PML, an inhibitorof eIF4E dependent cyclin D1 mRNA export (Cohen et al., 2001;Topisirovic et al., 2002), on the export of target mRNAs. We observeddecreased export (data not shown), and reduced protein levels of ODC,c-Myc, cyclin D1 and cyclin E1 mRNAs (data not shown) in cellsoverexpressing PML. Also, PML did not reduce levels of eIF4E, β-actin orGAPDH proteins (FIG. 18 c) and there was no alteration in total mRNAlevels for any of these transcripts when PML was overexpressed (data notshown). Thus, PML acts as an inhibitor of eIF4E dependent mRNA export,not just as an inhibitor of cyclin D1 mRNA export.

In summary, the physical association of mRNAs with the nuclear fractionof eIF4E is strongly correlated with their enhanced nuclear export. Inthe cytoplasm, these mRNAs may (i.e. ODC) or may not (i.e. cyclin D1) bea subject of modulation by eIF4E at the level of translation. Thus,eIF4E mediated modulation at the nuclear level neither precludes nornecessitates such modulation at the cytoplasmic level.

The RNA USER Code for eIF4E Dependent mRNA Export

Since we previously identified a 100 nucleotide eIF4E sensitivityelement (4E-SE) in the 3′ UTR of cyclin D1 which sensitizes cyclin D1and corresponding chimeric LacZ constructs to regulation by eIF4E at themRNA export level (Culjkovic et al., 2005), we carried out an extensivebioinformatics analysis to identify 4E-SE like elements in the othertarget RNAs identified in Table IV. Sequence analysis indicated that the4E-SE was well conserved in cyclin D1 transcript (from birds to humans)(Culjkovic et al., 2005), but comparison of cyclin D1 and the othereIF4E sensitive transcripts identified here failed to reveal any sharedsequence homology. We therefore examined the possibility that the 4E-SEelement is a structurally conserved element.

To best identify the common elements in the target mRNAs, we decided tocompare the cyclin D1 4E-SE with the 4E-SE from one of the newlyidentified target mRNAs, Pim-1 (we mapped the region of Pim-1 3′UTR tofunctional 4E-SE, FIG. 19 c). We mapped the 4E-SE from cyclin D1 and the4E-SE from Pim-1 to a minimal ˜50 nucleotide region (FIG. 19 a). Theseminimal domains, when fused to heterologous LacZ mRNA, immunoprecipitatewith eIF4E and have their mRNA export promoted by eIF4E (FIG. 19 c).Thus, we show that both of these minimal ˜50 nucleotide elements arefunctional 4E-SE. Although there was no sequence homology observed, bothelements contain two predicted adjacent stem loop pairs.

We used nuclease digestion methods to determine if this two functional4E-SEs had conserved secondary structural features, such as thepredicted stem loop structures. Importantly, these studies revealed thatboth elements fold into similar secondary structures. We refer to thiselement as adjacent stem loop pair (FIGS. 19 a and 19 b). Consistently,biophysical analysis indicates that Pim-1 and cyclin D1 4E-SEs havesimilar biophysical properties. For instance, circular dichroismanalysis of thermal melting curves using purified RNA oligomers forcyclin D1 and Pim-14E-SEs revealed multiphase behavior consistent withthe presence of multiple structural elements with different Tm's(Topisirovic et al., in preparation). Thus, both Pim-1 and cyclin D14E-SEs have similar secondary structures, consisting a two adjacent stemloop elements.

An initial problem we encountered in these studies is that the presenceof stem loop elements is common in the 3′ UTRs of cyclin D1 and Pim-1.In cyclin D1 alone, the PatSearch programme (Grillo et al., 2003)predicts ten potential stem loop structure pairs, and yet our previousstudies indicate that the only part of the cyclin D1 3′UTR that canimpart eIF4E sensitivity is the above defined 4E-SE (Culjkovic et al.,2005). Similarly, the Pim-1 3′ UTR contains two predicted adjacent stemloop pairs, while only one is a functional 4E-SE. Thus, we compared thesecondary structures of Pim-1 and cyclin D1 4E-SEs, to determinefeatures that would enable us to distinguish functional 4E-SEs fromother stem loop pairs. Visual inspection of the secondary structuresreveal the conservation of a set of A and U nucleotides (UX₂UX₂A,highlight in FIG. 19 a). Importantly, these patterns of nucleotides werenot found in any of the other stem loop pairs found in cyclin D1 orPim-1 3′UTR. Thus, these are features that can be used to distinguishfunctional 4E-SEs from other elements that have potential to fold intosimilar secondary structures.

Further analyses showed that the stem loop pair structure with theconserved pattern of nucleotides is also present in all of the othereIF4E sensitive targets identified here. Importantly, none of the mRNAsthat are not eIF4E sensitive contain stem loop pairs with the conservedpattern of nucleotides found in the functional 4E-SEs. In summary, wehave identified a structural motif, consisting of two adjacent stem looppairs, which impart eIF4E sensitivity. Importantly, there exist in thismotif sequence features of 4E-SEs that can be used to distinguishfunctional 4E-SEs from other paired stem loop structures.

The 4E-SE is Sufficient for Localization with eIF4E Nuclear Bodies

To assess whether the 4E-SE acted as an RNA zipcode for eIF4E nuclearbodies, LacZ chimeric constructs with either Pim-1 or cyclin D1 4E-SEwere expressed in U2OS cells. Both chimeric mRNAs co-localize with eIF4Enuclear bodies (FIG. 19 d). In the absence of the 4E-SE, no localizationof LacZ transcripts to eIF4E nuclear bodies is observed (FIG. 19 d).Importantly, LacZ-4E-SE does not associate with eIF4E bodies thatcontain the negative regulator, PML. This is consistent with ourprevious studies showing that there are two classes of eIF4E nuclearbodies: those that co-localize with PML and those that co-localize withendogenous cyclin D1 mRNA. Thus, endogenous cyclin D1 mRNAs co-localizewith eIF4E nuclear bodies that do not contain PML (Culjkovic et al.,2005). In this way, LacZ-4E-SE transcripts and endogenous mRNAs behavesimilarly.

These experiments demonstrate that the 4E-SE is sufficient to localizecapped mRNAs into eIF4E nuclear bodies irrespective of the rest of themRNA sequence. Moreover, the 4E-SE from Pim-1 and cyclin D1 arefunctionally equivalent in terms of localization activity. Thus, the4E-SE provides an RNA zipcode for localization to eIF4E nuclear bodies.

The 4E-SE Makes eIF4E Dependent Complexes

To establish whether the 4E-SE functions simply as a localizationsignal, or whether it acts in the formation of eIF4E dependent mRNPs, wecarried out EMSA assays. Studies were carried out with both theLacZ-cyclin D1-4E-SE (c4E-SE) and the LacZ-Pim-1-4E-SE (p4E-SE) toensure that assembly of these complexes is dependent on the 4E-SE itselfand not features specific to either 4E-SE. RNA probes were ³²P 3′ endlabeled and m⁷G capped. Addition of either mouse eIF4E with a 6 kDsolubility tag (m4E), or untagged human eIF4E (h4E) led to the formationof slower migrating species for both LacZ-4E-SE constructs (FIGS. 20 aand 20 b). Importantly, addition of nuclear lysates led to the formationof significantly higher molecular weight complexes, indicating thatproteins other than eIF4E are likely to be present. Complex sizes wereapproximately the same for both 4E-SE constructs. Addition of coldcompetitor 4E-SE RNAs led to a reduction in signal, consistent with the4E-SE element competing for the labeled 4E-SE containing transcripts(FIG. 20 e). Addition of nuclear lysates to LacZ transcripts lacking the4E-SE did not lead to formation of these complexes (FIG. 20 b).

To determine whether the 4E-SE complexes formed from nuclear lysateswere dependent on eIF4E, EMSA assays were performed with nuclear lysatesdepleted of eIF4E via immunoprecipitation. We estimated that lysateswere at least 80% depleted of eIF4E (data not shown). Lysatesimmunodepleted of eIF4E did not produce high molecular weight complexes(FIG. 20 b). Addition of purified tagged eIF4E to immunodepleted lysatesled to a partial restoration of the complex, which could be expected,since only eIF4E, but not other factors that were depleted during theanti-eIF4E immunoprecipitation, were re-introduced. Thus, eIF4E andassociated factors are required for formation of these RNPs. Inaddition, an antibody to eIF4E leads to a super shift of complexesformed from nuclear lysates (FIG. 20 b). Identical results are observedfor LacZ-p4E-SE. Finally, a mutant that disrupts the first stem loop(G₁₀C₁₁G₁₂ mutated to CAC) in the p4E-SE is defective in complexformation (FIG. 20 c). Thus, the 4E-SE element forms complexes dependenton eIF4E and on the structure of the 4E-SE.

To further characterize these complexes, LacZ-4E-SE constructs wereUV-cross-linked followed by RNase digestion and SDS-gel electrophoresis(FIG. 20 d). As for the EMSA studies, transcripts were m⁷G capped and 3′end labeled and the effects of addition of purified eIF4E or nuclearlysates to the size of cross-linked complexes was monitored. Since mRNAswere 3′ end labeled, binding of the cap only by purified eIF4E was notsufficient to protect the rest of the RNA from RNase digestion. Additionof the nuclear lysate leads to substantial shifts in molecular weight.Importantly, the LacZ-c4E-SE and the LacZ-p4E-SE form complexes similarin size. Three discrete species of between 75-90 kD are observed(indicated by arrows). The same complexes are absent in eIF4E depletednuclear lysate, indicating that these require eIF4E to form.Consistently, treatment of the nuclear lysate with the m⁷GpppG capanalogue (nc cap) also disrupts 75-90 kD range complexes. These speciesare absent from the LacZ controls, which lack the 4E-SE. A lower band,at ˜64 kD, is present in all the experiments, likely indicating theformation of some general RNP, not directly involved with eIF4E and the4E-SE. In summary, we observe two types of complexes: those ones thatcan form in the absence of eIF4E and are cap and 4E-SE independent (seeasterisk), and the second type that depends on eIF4E, the m⁷G cap and astructurally intact 4E-SE. The UV-cross-linking studies together withthe EMSA results indicate that the 4E-SE acts both as a zipcodelocalizing mRNAs to bodies (FIG. 19 d) as well as USER code for theeIF4E nuclear mRNP (FIG. 20).

eIF4E Dependent mRNA Export is Independent of On-Going Protein or RNASynthesis

We examined the importance of new protein synthesis and transcriptionfor eIF4E dependent mRNA export. To inhibit protein synthesis, cellswere treated with 100 μg/ml cycloheximide for 1 hour. (FIG. 21 a). Also,export of endogenous cyclin D1 mRNA was not modulated by cycloheximidetreatment (data not shown). Similarly, actinomycin D treatment (10μg/ml) did not affect export of these mRNAs (FIG. 21 a). Althoughcycloheximide treatment did not modify export, it is still possible thatthe 4E-SE could modulate polysomal loading in an eIF4E dependent manner.Thus, we monitored polysomal profiles of LacZ as a function of the 4E-SEand of eIF4E overexpression. The profiles of LacZ and LacZ-c4E-SE areindistinguishable and are not altered by eIF4E overexpression (data notshown). This is consistent with the finding that eIF4E overexpressiondoes not change cyclin D1 mRNA polysomal loading (Rousseau et al.,1996). Given that eIF4E dependent mRNA export is independent of on-goingprotein synthesis and that the 4E-SE does not alter polysomal loading,the functions of eIF4E in mRNA export and translation appear to bedecoupled.

We previously demonstrated that LacZ-c4E-SE transcripts did not havealtered stability relative to LacZ transcripts using actinomycin D overthe course of several hours (Culjkovic et al., 2005). However, it isstill possible that mRNA turnover could be substantially more rapid thanhours. Thus, we constructed LacZ and LacZ-4E-SE TetON-inducible celllines and examined the stability of these mRNAs immediately upondoxicycline addition. The presence of the 4E-SE does not substantiallyalter stability of the LacZ transcripts in either short (minutes) orlong term (hours) (data not shown).

eIF4E Dependent mRNA Export Pathway is Saturated by Excess 4E-SE

We reasoned that if the 4E-SE is required for export, overexpression ofLacZ-c4E-SE or LacZ-p4E-SE should specifically inhibit export of other(endogenous) 4E-SE containing mRNAs by competing for the 4E-SE specificexport machinery (FIGS. 21 b and 21 c). Using our TetON-inducible LacZ,LacZ-p4E-SE or LacZ-c4E-SE constructs, we monitored export of chimericmRNAs as a function of total mRNA levels. At early time points, whenlevels of LacZ mRNAs are low, 4E-SE export is more efficient with higherratios of cytoplasmic to nuclear chimeric mRNAs. As the levels of thesemRNAs increase, 4E-SE export becomes saturated and the ratio ofcytoplasmic to nuclear chimeric mRNAs decreases (FIG. 21 b). At the sametime, export of endogenous cyclin D1 mRNA was competed (impaired) byexpression of 4E-SE chimeric mRNAs (FIG. 21 b). Further, export of VEGFmRNA was not affected, consistent with its insensitivity to eIF4E at themRNA export level (FIG. 21 b). Thus, overexpression of the 4E-SE elementleads to competition for the 4E-SE specific export machinery.

4E-SE mediated Export is NXF1 Independent but CRM1 Dependent

Since the best-described cellular mRNA export pathway involves theNXF1/p15 heterodimer which appears to mediates bulk mRNA export (Cullen,2000; Cullen, 2003a), the dependence of the 4E-SE mRNA export on NXF1was examined (data not shown). Consistent with previous studies, as wellas our own, eIF4E does not immunoprecipitate with NXF1 in the nuclearfraction of cells ((Lejeune et al., 2002) and data not shown). However,this does not preclude a NXF1 dependent mechanism where eIF4E does notneed to physically associate with NXF1. To further investigate NXF1involvement in 4E-SE export, Flag tagged NXF1 or NXF1/p15 overexpressingcells were immunoprecipitated with anti-Flag antibodies and the presenceof LacZ or LacZ-c4E-SE mRNAs was monitored by real time PCR (FIG. 22 a).In contrast to LacZ mRNA that is enriched in the NXF1 fractions,LacZ-c4E-SE mRNA appears to be rather excluded. These results areindependent of the presence or absence of p15 (data not shown).

We extended these studies to examine the effects of knocking down NXF1expression on LacZ-c4E-SE export (FIG. 21 b). Overexpression of eIF4Eenhanced export of LacZ-c4E-SE transcripts, even when NXF1 levels weresubstantially reduced, indicating that export of LacZ-c4E-SE in thepresence of overexpressed eIF4E is independent of NXF1. In the absenceof the 4E-SE, LacZ mRNA cytoplasmic/nuclear ratio was substantiallyreduced by NXF1 depletion. Analysis of LacZ protein levels confirmed theabove findings (FIG. 22 c). As expected, siRNA treatment led toreduction in NXF1 levels whereas treatment with scrambled controls didnot (FIG. 22 c). Further, levels of eIF4G were not altered consistentwith studies which show longer siRNA treatments (>72 hours) are neededto reduce eIF4G levels (Herold et al., 2001). Thus, export of 4E-SEcontaining transcripts is independent of the NXF1 pathway. This does notrule out the possibility that a subset of 4E-SE transcripts do transitthrough this pathway, simply that they do not require this pathway to beexported.

Since many RNAs can be exported through the CRM1 pathway, we examinedthis possibility by using leptomycin B (LMB), a specific inhibitor ofCRM1 (Cullen, 2003a; Cullen, 2003b). The export of LacZ or LacZ-c4E-SEmRNAs as a function of overexpressed eIF4E and LMB treatment wasmonitored using real time PCR (data not shown). Strikingly, LMBsuppressed export of the LacZ-4E-SE constructs but not of LacZ orβ-actin transcripts. LMB leads to retention of 18S rRNA (FIG. 22 c),consistent with previous studies showing that ribosomal RNA exportrequires CRM1 (Moy and Silver, 2002).

Novel Export Pathway Involving eIF4E and 4E-SE Containing mRNAs

Since it was described, no underlying mechanism for eIF4E dependentexport has been determined (Rousseau et al., 1996). There are severalcharacteristic features that differentiate eIF4E mediated export fromthe pathway used for bulk mRNA (summarized in FIG. 23): 1) 4E-SEsaturates export of the eIF4E pathway but does not effect export of bulkmRNA (FIG. 21 b); 2) LMB inhibits eIF4E dependent export (FIG. 21 a); 3)the m⁷G cap is required for the eIF4E pathway (FIG. 18 b, Table V).Interestingly, there are many parallels between the eIF4E pathway andUsnRNA export: both are CRM1 dependent and both require the m⁷G cap.However, in contrast to the eIF4E pathway, UsnRNA export depends on RNAsbeing CBC bound in complex to PHAX, which acts as an adaptor for CRM1(Cullen, 2000; Cullen, 2003a; Cullen, 2003b; Ishigaki et al., 2001;Izaurralde et al., 1995).

In general, CRM1 mediated mRNA export requires co-factors which dependon the type of RNA being exported i.e. large rRNA, small rRNA, 5S rRNAor UsnRNA (Cullen, 2003a; Cullen, 2003b). Our previous studies indicatethat eIF4E overexpression does not modulate the export of 18S or 28SrRNA, which is CRM1 dependent, or tRNA, which is exported using theexportin-t receptor (Sarkar and Hopper, 1998). Thus, we hypothesize thateIF4E, or some subset of factors associated with the 4E-SE RNP, requireCRM1-adaptor proteins specific to the eIF4E dependent pathway. Further,these adaptors are found in limiting amounts and titratable by high4E-SE levels or by immunodepletion of eIF4E. Identifying such adaptorproteins will be an area of intense future work.

A conundrum in understanding eIF4E dependent mRNA export results fromthe observation that eIF4E stimulates the export of mRNAs that can bestill exported under physiological eIF4E levels. Thus, eIF4E dependentmRNA export is a means by which the cell rapidly upregulates geneexpression by stimulating the export of mRNAs that can be exportedthrough other pathways, albeit less efficiently. When eIF4E levels arelow, or in the absence of the m⁷G cap or 4E-SE, transcripts are exported(presumably) through the NXF1 pathway. This idea is consistent withprevious suggestions that the NXF1 pathway is a default mRNA exportpathway for those mRNAs that do not have any special features associatedwith them (Cullen, 2003b). In this way, eIF4E levels can act as a“cellular rheostat”. As levels increase, eIF4E sensitive mRNAs areexported much more efficiently, and in a coordinated fashion, throughthe eIF4E dependent CRM1 sensitive pathway described here. A recentreport indicates that CRM1 dependent mRNA export can occur during T cellactivation indicating that external cellular signals can lead toalterations in mRNA export pathways (Schutz et al., 2006).

The Role of eIF4E in 4E-SE Containing mRNA Export and Implications forCancer

The studies reported here suggest the possibility that the proliferativeand transforming properties associated with eIF4E are, at leastpartially, a result of dysregulation of eIF4E dependent mRNA export.These studies indicate a role for eIF4E in coordinating export andexpression of transcripts involved in cell cycle progression,proliferation and survival. Importantly, eIF4E does not promote theexpression of negative regulators of itself, i.e., PML. eIF4E alsopromotes the expression of c-Myc, a factor which upregulatestranscription of eIF4E in some cellular growth conditions (Schmidt,2004). Thus, eIF4E modulates the expression of many genes involved inmultiple points of cell cycle progression.

The 4E-SE provides a USER code for targeting these transcripts forexport in an eIF4E sensitive manner. Other transcripts may be regulatedby eIF4E at translation level using USER codes different from the 4E-SE.Further, the 4E-SE may associate with other, as yet to be identifiedRNPs. In this way, the effects of eIF4E and regulation of 4E-SEcontaining transcripts are likely to be complex and combinatorial. Forinstance, translation of export sensitive mRNAs does not depend on the4E-SE, but rather the complexity of the 5′UTR. Transcripts such as Pim-1and ODC (Hoover et al., 1997; Rousseau et al., 1996) serve as examplesof the combinatorial use of USER codes for modulating gene expressionand support the idea of the use of such a network. Consistently, ourstudies indicate that the translation and export functions of eIF4E canbe decoupled based on the composition of the 3′ and 5′ UTRs (i.e. eIF4Eenhances the export of cyclin D1, but enhances the translation of VEGF).

Several key regulators of eIF4E dependent mRNA export have beenidentified including PML (Cohen et al., 2001) and several homeodomainproteins which contain conserved eIF4E binding sites (Topisirovic etal., 2003a; Topisirovic et al., 2005). These regulators are positionedto modulate the entire RNA regulon, potently modulating cell cycleprogression and cell survival. Our studies demonstrate that PML and PRHimpede eIF4E dependent export of cyclin D1 and other 4E-SE containingtranscripts ((Culjkovic et al., 2005; Topisirovic et al., 2003a) andthis report). Stimulators of this growth regulon include HOXA9, whichpromotes both the mRNA export and translation of genes in the regulon(Topisirovic et al., 2005). The far-reaching activities of theseregulators, particularly those that regulate multiple eIF4E functionssimultaneously, likely lies in their ability to modulate eIF4E, a keynexus in this regulon.

The physiological importance of this regulation is clear. In primaryspecimens from acute myeloid leukemia patients, PRH is bothdownregulated and de-localized from eIF4E nuclear bodies (Topisirovic etal., 2003b). At the same time, HOXA9 is upregulated and becomesassociated with eIF4E in both the nuclear and cytoplasmic compartments,leading to upregulation of both eIF4E dependent mRNA export andtranslation (Topisirovic et al., 2005).

In conclusion, eIF4E dependent modulation of mRNA export could providean immediate response system by which the cell responds to extracellular stimuli prior to transcriptional reprogramming. Our resultsindicate that modulation of mRNA export allows coordinated modulation ofcellular proliferation, and provide one of the first examples of an RNAregulon that is positioned to directly impact on human disease. Abilityof eIF4E to modulate coordinated gene expression impacting onproliferation and cell survival pathways ensures maximum efficiency forits growth promoting potential. Certainly, these findings do notpreclude, but rather complement, critical modulation of gene expressionby eIF4E at other levels of mRNA metabolism, in particular translationand mRNA stability/sequestration. In summary, we define a novel mRNAexport pathway, which is used for coordinate expression of genes thatgovern cell cycle progression and survival.

RNA Regulons as Therapeutic Targets

Identification of nodes in networks clearly defines such nodes aspositions in the cellular gene expression circuitry which could bepotent drug targets (FIG. 24).

Recent studies have focused on the mTOR inhibitor, rapamycin, as a meansto downregulate protein synthesis in part by inhibiting phosphorylationof the eIF4E binding proteins (eIF4Ebp or 4E-BP) and thereby allowingthese to interact and inhibit eIF4E activity in the cytoplasm(Sarbassov, D. D., Ali, S. M. & Sabatini, D. M. (2005) Curr Opin CellBiol 17:596-603). However, treatment with rapamycin in these patientshas led to increased levels of activated Akt in their tumours whichsupport clinical data indicating that rapamycin may be of limited use asa single agent cancer therapy (O'Reilly, K. E. et al. (2006) Cancer Res66: 1500-8. Furthermore, cells overexpressing eIF4E show increasedresistance to rapamycin and the combination of this drug withdoxorubicin (Wendel, H. G. et al. (2006) Cancer Res 66, 7639-46).Ribavirin provides an alternative strategy to target eIF4E activity asit directly targets eIF4E via its mimicry of the m⁷G cap (Kentsis, A.,et al. (2004) Proc Natl Acad Sci USA; Kentsis, A. et al. RNA 11:1762-6). Thus, it is positioned to efficiently shut down the eIF4Eregulon.

TABLE IV List of mRNAs that associate (or not) with nuclear eIF4E.Function/Growth Translationally Target RNA Promoting propertiesSensitive to eIF4E Cyclin D1 G1/S cell cycle progression no (Rousseau(gi: 77628152) (Liang and Slingerland, 2003) et al., 1996) Cyclin E1G1/S cell cycle progression n.d. (gi: 17318558) (Liang and Slingerland,2003) Cyclin A2 S/G2/M cell cycle progression n.d. (gi: 16950653) (Liangand Slingerland, 2003) Cyclin B1 G2/M cell cycle progression yes (Caoand (gi: 34304372) (Liang and Slingerland, 2003) Richter, 2002) ODCPolyamine synthesis/tumor yes (Clemens and (gi: 4505488) promoting(Pegg, 2006) Bommer, 1999) Pim-1 S/T kinase (Bachmann and yes (Clemensand (gi: 31543400) Moroy, 2005) Bommer, 1999) Mdm2 Survival/apoptoticrescue n.d. (gi: 46488903) (Liang and Slingerland, 2003) c-MycFacilitates G1/S progression yes (Clemens and (gi: 71774082)Transcriptionally upregulates Bommer, 1999) eIF4E (Liang andSlingerland, 2003; Schmidt, 2004) Nibrin/NBS1 DNA repair/Akt activation/n.d. (gi: 67189763) promotes growth (Chen et al., 2005) Fbox1 Promotescell cycle n.d. (gi: 16306583) progression (Liang and Slingerland, 2003)CGGbp1 Influences FMR1 expression n.d. (gi: 56550052) (Naumann et al.,2004) P54nrb/NONO.1 RNA binding protein/Promotes n.d. (gi: 34932413)survival (Stier et al., 2005) Selenoprotein S Glucose regulated ERprotein n.d. (gi: 45439347) (Gao et al., 2004) GAPDHHousekeeping/apoptotic no (Clemens and (gi: 83641890) Bommer, 1999;Rousseau et al., 1996) VEGF Mitogen/Angiogenesis/tumor yes (Clemens and(gi:71051577) invasion (Roy et al., 2006) Bommer, 1999) P53Pro-apoptotic/reduces eIF4E no (Clemens and (gi: 8400737) transcription(Zhu et al., Bommer, 1999) 2005) β-actin Cytoskeletal no (Rousseau et(gi: 5016088) al., 1996) α-tubulin Cytoskeletal no (gi: 57013275) eIF4ETranslation and mRNA export/ no (Clemens and (gi: 54873625) Promotesgrowth and survival Bommer, 1999; (Strudwick and Borden, 2002) Strudwickand Borden, 2002) PML Pro-apoptotic/G1 arrest no (Strudwick and (gi:67089161) (Borden, 2002) Borden, 2002) α-globin Housekeeping No (gi:14456711) c-ebpa Arrests proliferation n.d. (gi: 28872793) (Wang et al.,2001) n.d. not determined.

TABLE V Cytoplasmic/nuclear ratio of different mRNAs in U937 cellsoverexpressing eIF4E wt or mutants. MRNA MSCV vector ctrl 4Ewt W73A W56ACyclin D1 1 ± 0.035 11.995 ± 0.860  11.450 ± 860   1.110 ± 0.036 CyclinE1 1 ± 0.022 3.442 ± 0.05  3.172 ± 0.208 1.200 ± 0.015 Cyclin A2 1 ±0.044 5.472 ± 0.580 7.736 ± 0.540 1.002 ± 0.058 Cyclin B1 1 ± 0.1084.720 ± 0.750 4.073 ± 0.434 1.475 ± 0.122 ODC 1 ± 0.010 6.847 ± 0.3737.138 ± 0.852 1.272 ± 0.018 Pim-1 1 ± 0.051 3.435 ± 0.194 3.391 ± 0.2511.029 ± 0.029 Mdm2 1 ± 0.325 15.698 ± 0.160  15.097 ± 0.793  1.379 ±0.014 c-Myc 1 ± 0.033 2.980 ± 0.233 2.857 ± 0.226 0.925 ± 0.042 Nibrin 1± 0.030 4.728 ± 0.145 4.958 ± 0.230 1.226 ± 0.024 F-box 1 1 ± 0.06911.202 ± 0.866  10.713 ± 0.633  1.363 ± 0.062 Selenoprotein S 1 ± 0.07214.520 ± 1.164  11.839 ± 0.257  1.193 ± 0.234 VEGF 1 ± 0.111 0.835 ±0.063 0.980 ± 0.261 1.387 ± 0.022 β-Actin 1 ± 0.173 1.020 ± 0.238 1.220± 0.203 1.313 ± 0.180 P53 1 ± 0.016 0.892 ± 0.006 1.392 ± 0.230 0.994 ±0.008 α-globin 1 ± 0.379 0.861 ± 0.237 1.265 ± 0.232 1.275 ± 0.346Cytoplasmic/nuclear (n/c) values represent relative fold ± sd,normalized to vector control (MSCV), which was set to 1. Average values± sd were calculated for each set of triplicates. Average values of allanalyzed mRNAs obtained for each fraction of each sample were divided byGAPDH mRNA values obtained for the same fraction/sample. After dividingcytoplasmic with nuclear values of each sample, obtained c/n values ± sdwere normalized by setting MSCV vector control c/n value to 1.

Example 5 Pre-Clinical and Clinical Evaluation of Ribavirin as a NovelTherapy for Breast Cancer

Breast cancer is an important and biologically complex human diseasewhich is newly diagnosed in 200,000 individuals per year and accountsfor over 40,000 deaths annually within the United States. Over the pastseveral decades clinicians have come to realize that breast cancer isnot a single uniform disease but one that can be segmented intodifferent clinical subtypes. Until recently subtype designations werelargely clinically based, but with the introduction of multiplexed geneexpression and tissue microarray technologies our capacity to identifynovel clinical subtypes within the overall breast cancer milieu has beensignificantly enhanced. For the purposes of this Example, breast cancerswill be considered to belong to discrete clinical subtypes based upon aset of widely employed biomarkers [estrogen receptor (ER), progesteronereceptor (PR), erbB2/neu/HER2 receptor (HER2)] and/or their pathologicgrade (I to III). These attributes, together with age, tumor size andaxillary lymph node status, provide important prognostic and predictiveinputs into the clinical management of breast cancer. For example, ER/PRpositive status correlates with enhanced short-term prognosis and ispredictive for response to tamoxifen therapy, while HER2 amplificationor overexpression correlates with increased recurrence rates, tumoraggressiveness, increased mortality in node-positive patients and is apositive predictor of response to trastuzumab. By contrast, breastcancers belonging to the basal-like subtype (aka ‘triple negative’ sincethey lack all three ER/PR/HER2 receptor biomarkers) are considered to bedistinct from all others classes and generally have a poor prognosis.Despite these defined differences between subtypes, high-grade tumors(poor prognosis) exist in all three major classes including: ER/PRpositive (ca. 15% all breast cancers), HER2 positive (ca. 20% all breastcancers) and basal-like (ca. 20% all breast cancers).

Development of effective therapeutic strategies for breast cancers witha poor prognosis (basal-like, HER2-positive and hi-grade tumors)represents an important unmet medical need. Anthracyclines, taxanes,trastuzubab (monoclonal antibody against the extracellular domain ofHER2) and bevacizumab (anti-vascular endothelial growth factormonoclonal antibody) and combinations thereof represent many of theestablished and/or investigational therapies available for the treatmentof breast cancer. Even when clinically effective their use is oftenassociated with significant cardiac and neuronal toxicities, neutropeniaand even gastrointestinal perforation and hemorrhaging in the case ofbevacizumab. Development of effective breast cancer therapies withlimited/no associated toxicity is another important unmet medical need.

In human malignancies, including breast cancer, eIF4E overexpressioncorrelates with enhanced metastatic potential and an overall poorclinical prognosis. Relevant to breast cancer is the finding that eIF4Elevels are significantly increased in the vascularized malignant ductsof invasive carcinomas and that within the surgical margins of breastcancer patients elevated levels of eIF4E correlate with an increasedrate of disease recurrence that is independent of nodal status.Recently, Li and co-workers determined that elevated eIF4E levels inbreast cancers correlate with higher VEGF levels and increasedmicrovessel density. Further, eIF4E-dependent activation of thetranslation initiation complex eIF4F has been determined to be essentialfor the genesis and maintenance of the malignant phenotype in humanmammary epithelial cells. Increased eIF4E activity plays an importantprognostic role in human breast cancer.

Critical to our overall hypothesis that eIF4E activity is elevated inbreast cancers will be our identification of which clinical subtype(s)possess elevated levels of eIF4E. Multiplexed gene expression and tissuemicroarray analyses will permit us to identify breast cancer eIF4Esubytpes. To date, direct correlation to other clinical subtypes on apatient by patient based is not available.

A breast cancer cohort analyzed consists of 688 samples of invasiveductal carcinoma selected from the Yale University Department ofPathology archives as available from 1961 to 1983 with approximatelyhalf node-positive specimens and half node-negative specimens. Analysisperformed using a HistoRx's robust method of objective in situquantification of protein expression that employs immunofluorescencestaining and fluorescence microscopy technology (AQUA™ analysis) thatwas originally developed by Dr. Robert Camp and Dr. David Rimm at YaleUniversity. The AQUA™ system allows for high-throughput, quantitativehigh resolution analysis of tissue microarrays, whole tissue sectionsand core biopsy samples of human tissues, animal tissues, xenografts andcell lines. In contrast to many automated imaging methods, AQUA™analysis is not morphology based, but rather is based on molecularco-localization of different fluorophores tagged to different antibody-or stain-defined compartments. By use of two different analysisalgorithms, the resulting AQUA™ scores are objective and areproportional to the concentration per unit area on a continuous scaleequivalent to an ELISA, while maintaining critical spatial informationof tissue samples.

Expression of eIF4E was assessed by AQUA using the Epitomics anti-eIF4Eantibody at a dilution of 1:500. eIF4E expression was compartmentalizedusing cytokeratin as a tumor marker and DAPI as a nuclear marker.Appropriate images were captured using the PM1000. AQUA was validatedfor compartmentalization of expression. AQUA scores were linked toclinical data and analyzed. Unsupervised hierarchical clusteringanalysis (FIG. 25, average linkage of mean-based Z-scores) was performedusing AQUA scores for estrogen receptor (ER), progesterone receptor(PR), epidermal growth factor receptor (EGFR), Her2, and eIF4E. HigheIF4E clustered predominantly with the basal (triple-negativetumors—ER/PR/Her2 negative) phenotype—Defined herein as BASAL/eIF4E+.Not all basal-like tumors showed elevated eIF4E expression, suggestingthat eIF4E may be a novel biomarker defining a specific sub-populationof basal-like tumors. While eIF4E expression is clearly most closelylinked a particular Basal-like sub-phenotype, it also associates to alesser extent with the Her2+ molecular subtype and the ER+/PR− molecularsubtype.

FIG. 25 depicts unsupervised hierarchical clustering analysis of proteinexpression level in breast cancers performed using AQUA scores forestrogen receptor (ER), progesterone receptor (PR), epidermal growthfactor receptor (EGFR), Her2, and 4E. FIG. 26 depicts expressionanalysis performed from bulk tumor RNA extracted from 141 primary breastcancers and run on Affymetrix U133 plus 2.0 arrays (Andrea RichardsonDana-Farber Cancer Institute). The analysis was performed usinghierarchical clustering function of dChip software. Comparison of high4E expressors to low expressor tumors one finds that 4E isco-overexpressed with a set of genes from chromosome 4q 21-31 (indicatedby black bar on right), and co-overexpressed with other cell-cycleproliferation genes.

High 4E clustered predominantly with the basal (triple-negativetumors—ER/PR/Her2 negative) phenotype (“BASAL/4E+”). Not all basal-liketumors showed elevated 4E expression, suggesting that 4E may be a novelbiomarker defining a specific sub-population of basal-like tumors. While4E expression is clearly most closely linked a particular basal-likesub-phenotype, it also associates to a lesser extent with the Her2+molecular subtype and the ER+/PR− molecular subtype. Based upon ourpreliminary gene expression and AQUA analyses we conclude that theclinical inhibition of eIF4E activity (by Ribavirin) may demonstrate thelargest benefit in patients presenting with basal-like/eIF4E+breastcancers and selected HER2/eIF4E (+/+) and ER/eIF4E (+/+) cancers.Therefore, we will focus our initial pre-clinical and clinicaldevelopment efforts on Basal/eIF4E(+), HER2(+)/eIF4E(+) breast cancerclinical subtypes and to a lesser extent due to its more limitedassociation to ER(+)/eIF4E(+) breast cancers. The identification ofeIF4E clinical subtypes will serve to focus our pre-clinical andclinical studies and the detection methodologies will insure that we candetect changes within human clinical samples.

Breast cancer cell lines corresponding to established clinical subtypeswill be obtained and evaluated for eIF4E activity using the same set ofstudy attributes used to characterize human clinical samples in ourexpression and tissue microarray cohorts, namely: eIF4E protein level,eIF4E phosphorylation status, eIF4E-BP1 level and its phosphorylationstatus. The following cell lines are available for study and have beencharacterized with regards to their biomarker status by expressionprofiling (D. Iglehart, Dana-Faber Cancer Institute):

Basal-like HER2+/ER+ HER2+/ER− HER2−/ER+ HER2−/ER− EGFR BT474 (x) SKBR3(x) MCF-7(x)(4E) BT20, HCC38 MDA231(x)(4E) MDA361 MDA 453 T47d (x)HCC1187, HCC1143 MDA468(x) HCC1599, HCC1183 HCC1954 Code: (x) = Grows asxenograft; (4E) = Elevated eIF4E; Absence of label = To be determined

Breast cancer cell lines determined to possess elevated eIF4E activitywill be selected for further study with preference being given to celllines corresponding to human clinical subtypes determined to possesselevated eIF4E activity. Based upon our preliminary results we willfocus our efforts on Basal, HER2+/ER− and ER+/HER− cell lines.

Depending upon the cell line under consideration our analysis of eIF4Eactivity will be performed cells cultured under a variety of conditionsincluding: +/− serum, +/− hormone-stripped serum medium, and heregulinor EGF for cell lines expressing EGFR and HER2.

We will examine the ability of Ribavirin to inhibit in a dose-dependentfashion eIF4E functions at several levels. Specifically, we will examinethe impact of Ribavirin on (i) eIF4E-dependent mRNA transport andtranslation of select mRNAs and (ii) nuclear/cytoplasmic distribution ofeIF4E and select mRNAs. Select mRNAs will include at least cyclin D1,VEGF, FGF2 and as warranted HER2 and EGFR. Controls will useeIF4E-insensitive mRNA (housekeeping gene such as actin and GAPDH).

Lastly, we will examine the impact of Ribavirin on colony formation,cellular proliferation and induction of apoptosis using normal andbreast cancer cells lines using established methodologies.

Cell lines corresponding to human clinical subtypes possessing elevatedlevels of eIF4E protein (basal-like, HER2+, ER+ subtypes per ourpreliminary results) will be vetted for use in xenograft studies. Weanticipate examining cell lines determined to utilize any/all of themore subtle mechanisms of providing for elevated eIF4E activity, namelyeIF4E phosphorylation; eIF4E-BP1 levels and phosphorylation; or forexample subtypes with distinctive changes in nuclear:cytoplasmiclocalization especially if they are found to correlate with additionalnovel eIF4E breast cancer clinical subtypes.

We will also examine whether Ribavirin decreases the resistance ofbreast cancer cell lines to classical chemotherapeutic agents and/ortaxanes as might be predicted given the fact that we would anticipateRibavirin inhibiting eIF4E's anti-apoptotic activity, especially in thepresence and absence of FGF2.

In addition to studying the synergistic effects of Ribavirin onclassical breast cancer chemotherapeutic agents, we will investigatewhether Ribavirin acts synergistically with targeted agents [i.e.inhibitors of HER2, ER and EGFR and/or intracellular kinase activitiesincluding NFkB (using the specific IKK peptide inhibitor NBD) andPJ3K/Akt (using the specific PI3K inhibitor Ly294002)]. Breast cancercell lines will be obtained from ATCC or from the laboratory of D.Iglehart (Dana-Farber). Cells will be grown using established cultureconditions. Western (protein) analyses will employ commerciallyavailable antibodies against eIF4E, phospho-eIF4E, eIF4E-BP1 andphospho-eIF4E-BP1 and appropriate positive and negative controls.Similarly (mRNA) analyses will employ commercially available RNAextraction and mRNA probes. Required growth factors, chemotherapeuticagents and kinase inhibitors are commercially available and cell cycleanalysis and apoptosis analyses will be performed using establishedmethods and analyzed by fluorescent activated cell sorting.

Ribavirin's activity against breast cancers clinical subtypes will bedetermined in vivo using a mouse xenograft tumor model system and celllines vetted corresponding to the human breast eIF4E clinical subtypesidentified above. Specifically, we will examine Ribavirin's effect ontumor growth and protein expression of eIF4E sensitive molecules (atminimum FGF2, VEGF, cyclin D1 and Her2/neu or EGFR as warranted). Use ofartificial gene reporter constructs containing 3′UTR and/or 5′UTR mRNAcontrol structures coupled to an ‘internal’ reporter gene (LacZ, GFP)will enhance our ability to visualize Ribavirin eIF4E-sensitive mRNAs invivo. When feasible tumors will evaluated for apoptosis status. Andcontrol proteins and mRNAs to be analyzed will include housekeepinggenes (actin and GADPH).

Initially we will confirm that xenografts can be established uniformlyusing the human breast cancer cell line(s) of interest. If xenografts donot take uniformly we will then ‘passage’ tumors from animals wheretumors take as this as been proven as one way to facilitate theestablishment of tumor xenografts. Alternatively we will implant humanbreast cancer cells in the presence of matrigel which has also provenuseful in xenograft model systems (Iglehart, personal communication).

Once appropriate xenograft models corresponding to eIF4E-relevant humanclinical subtypes are established it will be important to establish thedose relationship between the concentration of Ribavirin administered toanimals and the impact on tumor growth. Control animals will be givenvehicle control. Our initial focus will be on vetting breast cancerxenograft models corresponding to human clinical subtypes possessingelevated levels of eIF4E protein (basal-like, HER2+, ER+ per ourpreliminary results) for use in xenograft studies. It is further ourintention to examine breast cancer xenograft models corresponding toemploy any/all of the more subtle mechanisms of providing for elevatedeIF4E activity, namely eIF4E phosphorylation; eIF4E-BP1 levels andphosphorylation; or for example subtypes with distinctive changes innuclear:cytoplasmic localization), in particular should these lines befound to correspond to novel eIF4E breast cancer clinical subtypes thatwe identify above. We will also examine whether Ribavirin decreases theresistance of breast cancer xenograft tumors to classicalchemotherapeutic agents and/or taxanes, if our in vitro data supportsthis line of investigation. Lastly, in addition to studying thesynergistic effects of Ribavirin on classical breast cancerchemotherapeutic agents, we will investigate whether Ribavirin actssynergistically with targeted agents [i.e. inhibitors of HER2, ER andEGFR and/or intracellular kinase activities including NFkB (using thespecific IKK peptide inhibitor) and P13K/Akt (using for example thespecific PI3K inhibitor Ly294002)] using appropriate eIF4E-based breastcancer xenograft model systems. These investigations will be driven byour analysis of eIF4E-activity based classification of human breastcancers using tissue microarray and gene expression analysis.

Nude mice (‘athymic’) will be obtained from The Jackson Laboratory.Other reagents and supplies required have been described above.

We hypothesize that strategies targeting eIF4E in patients with cancerscharacterized by elevated eIF4E levels is indicated. Ribavirin is awell-characterized, orally available, anti-viral drug. Ribavirin hasbeen determined to physically mimic the m7G mRNA cap structure and toinhibit thereby eIF4E activity and function. We propose to studyRibavirin as a novel and targeted treatment of patients with breastcancers characterized as having elevated levels of eIF4E activity.

Example 6 4E Modulates the Akt Pathway for its Survival Function

In this example we demonstrate that a small molecule can be used toinhibit a RNA regulon. Specifically, we demonstrate that ribavirin,through its inhibitory actions on eIF4E, can inhibit Akt survivalsignalling. In this way, ribavirin impedes both activation of Akt andproduction of downstream effectors of Akt.

Abstract

The coordinated regulation of post-transcriptional events is a means bywhich to modulate physiological processes. We postulate that theeukaryotic translation initiation factor, 4E, modulates gene expression,and thus biological activities, via coordinate mRNA export andtranslation of a subset of mRNAs. 4E not only promotes proliferation,but also rescues cells from apoptotic stimuli. Here we examine themolecular basis for 4E mediated apoptotic rescue of serum deprivedfibroblasts. First, we demonstrate that 4E overexpression leads toenhanced survival signaling by leading to activation of Akt. Next, wedemonstrate that 4E requires the presence of Akt1 in order to rescuefibroblasts. Further, we show that a mutant form of 4E, W73A 4E, rescuescells as readily as wildtype 4E. This mutant is active in promoting themRNA export function of 4E but not its translation activity. We showthat 4E mediates Akt activation through the upregulation of NBS1, afactor known to activate the PI3K/Akt pathway. 4E modulates theexpression of NBS1 at the level of mRNA export, and requires NBS1 toactivate Akt and to rescue cells from apoptosis. Further, 4Ecoordinately upregulates the expression of downstream effectors of theAkt pathway thereby amplifying the effects of Akt. The promyelocyticleukemia protein PML, a known regulator of 4E, inhibits 4E mediatedincreases in NBS1 export, in Akt activation and in apoptotic rescue.These studies provide a molecular basis for 4E mediated apoptoticrescue.

Introduction

Elevated levels of the eukaryotic translation initiation factor 4E (4E)are associated with oncogenic transformation in cell culture,tumorigenesis in mouse models and with poor prognosis in a significantsubset of human cancers (Graff and Zimmer 2003). 4E promotesproliferation and rescues cells from a variety of apoptotic stimuliincluding serum deprivation (Polunovsky et al. 1996; Sonenberg andGingras 1998; Tan et al. 2000; Graff and Zimmer 2003). At the molecularlevel, 4E modulates gene expression at two distinct levels: mRNAtranslation and mRNA nuclear export (Sonenberg and Gingras 1998;Culjkovic et al. 2005, Culjkovic et al., 2006; Culjkovic et al. 2007).To act in either of these processes, 4E must bind the m7G cap moietyfound on the 5′ end of mRNAs (Sonenberg and Gingras 1998; Culjkovic etal. 2005, Culjkovic et al., 2006; Culjkovic et al. 2007). In thecytoplasm, 4E recruits mRNA to the ribosome as a critical step intranslation initiation (Sonenberg and Gingras 1998). Not all transcriptsare affected equally by 4E (Sonenberg and Gingras 1998; Culjkovic et al.2005, Culjkovic et al., 2006; Culjkovic et al. 2007; Mamane et al.2007). For instance, the translation of a subset of genes with complex5′ UTRs is more sensitive to 4E levels (and are deemed 4E sensitive)than transcripts with short, unstructured UTRs. In this case,translation enhancement is defined as the association of thesetranscripts with heavier polysomes. In the nucleus, 4E upregulates themRNA export of a substantial subset of growth promoting mRNAs whichcontain a 50 nucleotide element known as the 4E sensitivity element(4E-SE) in their 3′UTR (Rousseau et al. 1996; Culjkovic et al. 2005,Culjkovic et al., 2006; Culjkovic et al. 2007). Increased proteinproduction of the corresponding export sensitive mRNAs arises throughtwo mechanisms: 1. a concentration effect whereby the increased levelsof cytoplasmic transcripts means that more protein is made, and/or 2. asubset of these transcripts are subsequently loaded more efficientlyonto the heavier polysomes because they are translationally sensitive to4E (Culjkovic et al. 2005, Culjkovic et al., 2006; Culjkovic et al.2007). Thus, 4E effects gene expression at multiple levels.

Recent studies indicate that there is a poor correlation between theproteomes and transcriptomes of cells (Lu et al. 2006). This impliesthat post-transcriptional regulation plays a critical role in geneexpression and thereby impacts on the resulting physiology of the cell.Keene and colleagues proposed the RNA regulon model to describe a meansby which post-transcriptional gene regulation can be coordinated(Tenenbaum et al. 2000; Keene and Tenenbaum 2002; Keene and Lager 2005).In this model, the expression of transcripts that act in the samebiological pathway, such as cell cycle progression, is coordinatelycontrolled by the presence of elements in the 3′ or 5′ UTR of thesemRNAs. These RNA elements are referred to as USER codes. For example, aset of mRNAs which encode proteins involved in the same biochemicalpathway would have their mRNA export coordinated by having a common USERcode in their 3′UTR, such as the 4E-SE. The USER codes work byrecruiting proteins involved in a given process to the RNAs in question,in order to facilitate said process. Thus mRNAs containing the 4E-SEUSER code would recruit the appropriate export factors to the RNAs,facilitating the export process. Any level of RNA metabolism could bemodulated this way, as long as the appropriate USER codes were present.

Our studies strongly suggest that 4E is a node in an RNA regulongoverning cell cycle progression by (at least in part) the combinatorialmodulation of the export of a wide variety of transcripts involved innearly every step of the cell cycle. Similarly, translationallysensitive mRNAs likely contain USER codes in their 5′ UTR (Mamane et al.2007). In this way, control at the mRNA export and translation levelscan be decoupled i.e. mRNAs would require both the 3′ 4E-SE and the 5′USER code in order to be modulated by 4E at both levels. This model ofgene expression provides network level control of the fate of mRNAs thatencode proteins involved in the same biochemical and thus biologicalprocesses. Control of nodes (such as 4E) in these regulons is criticalfor determining the fate of the cell.

Here, we examine the possibility that 4E rescues cells by coordinatelyregulating the expression of factors in such networks in order toachieve cell survival. We demonstrate that 4E potentiates Akt activationand that this activity is required for its ability to rescue cells fromserum deprivation induced apoptosis. The RNA regulon model serves as atheoretical context to understand how 4E coordinately, and potently,activates the Akt signaling pathway. First, 4E overexpression leads tothe upregulation of the expression of NBS1, a factor which mediatesphosphorylation of Akt. Second, 4E overexpression leads to thecoordinated mRNA export and thereby upregulation of several downstreameffectors of Akt. Further, a cellular inhibitor of 4E, the promyelocyticleukemia protein PML, inhibits 4E dependent Akt activation and alsoreduces the expression of a subset of downstream effectors of Akt. Inthis way, 4E and PML coordinately modulate an RNA regulon which controlsthe Akt pathway and thus potently modulates cell survival.

Results

4E Overexpression Promotes Akt Activation

We examined the possibility that 4E overexpression could lead toactivation of Akt. Akt activation was assessed by monitoring itsphosphorylation at T308 and S473 using phospho-specific antibodies andwestern analysis of immortalized murine embryonic fibroblasts (MEFs).Phosphorylation of these two sites is a well-characterized indicator ofAkt activation (Alessi et al. 1996; Alessi et al. 1997; Stokoe et al.1997; Nicholson and Anderson 2002; Vivanco and Sawyers 2002; Song et al.2005). Clearly, 4E overexpression leads to increased phosphorylation ofAkt at both sites (FIG. 27A). Importantly, the m7G cap binding mutant of4E (W56A), which is unable to act in translation or mRNA export(Culjkovic et al. 2005, Culjkovic et al., 2006), does not have thiseffect. In contrast, an 4E mutant (W73A) that acts in mRNA export butdoes not promote translation of sensitive mRNAs (Culjkovic et al. 2005,Culjkovic et al., 2006), also activates Akt. Consistent with Aktactivation, 4E and W73A mutant overexpression lead to enhancedphosphorylation of S6 and BP1, whereas the inactive W56A mutant does not(FIG. 27A). In terms of these activities, similar experiments in othercell types (NIH 3T3, U2Os and U937s) showed the same pattern of results(data not shown). Importantly, overexpression of wildtype or mutantforms of 4E did not lead to modulation of total Akt levels.

For comparison, we examined the effects of 4E overexpression in Akt1−/−cells (FIG. 27A and data not shown). For these studies, we used a cellline derived from MEFs (Akt1−/− cells) in which Akt1, the prevalent Aktform, was knocked out. The wildtype fibroblasts used above are thelittermate controls for these Akt1−/− cells. The Akt antibody usedrecognizes all three isoforms of Akt, and thus, one observes theseisoforms in the Akt1−/− cells (FIG. 27A).

Clearly, 4E does not induce phosphorylation of Akt1 due to the knockoutof this protein. Interestingly, there is more phosphoBP1 in general, inAkt wildtype versus Akt1−/− cells. This was observed previously and islikely due to the fact that the loss of Akt leads to reduced BP1phosphorylation as expected. Similar results were observed forphospho-S6. Further, there is no alteration in total levels of BP1 or S6in knockout cells relative to vector controls. Interestingly, 4E stillelevates BP1 and S6 phosphorylation in the knockout cells (withoutchanging total levels of either protein), suggesting that 4E can useAkt2 or Akt3 to activate mTOR and thereby lead to phosphorylation ofthese proteins (Easton et al. 2005; Skeen et al. 2006).

We tested whether 4E mediated Akt activation occurred in a PI3Kdependent manner. In cells overexpressing 4E, there is clearly morephosphorylation of Akt at both T308 and S473 relative to vectorcontrols. However, treatment of 4E overexpressing cells with the PI3Kinhibitor, LY294002 (Yao and Cooper 1995), led to a drastic reduction inphosphorylation of Akt at both sites (FIG. 27B), while Akt proteinlevels were not altered (FIGS. 27A and 27B). Further, treatment did notimpede the 4E dependent increases in NBS1, an 4E dependent mRNA exporttarget (Culjkovic et al. 2006). Thus, LY294002 did not alter this 4Eactivity. As expected, LY294002 inhibited phosphorylation of S6 and BP1(Sanchez-Margalet et al. 1994; Gingras et al. 1998). The potentialimplications of 4E modulation of BP1 phosphorylation are addressed inthe Discussion.

4E Requires Akt1 for its Survival Functions

We examined the relevance of Akt activation to 4E's establishedphysiological effects in cell survival. The ability of 4E to rescuewildtype or Akt1−/− cells from serum deprivation induced apoptosis wasmonitored using annexin V/propidium iodide staining in conjunction withflow cytometry and separately, TUNEL analysis (FIG. 28, data not shown).For comparison, cells that were not serum deprived are also shown.Importantly, 4E overexpression rescued wildtype cells (˜80% viablecells) versus vector controls (˜40% viable cells). The extent of rescueis similar to those shown in the original report describing the survivalfunction of 4E (Polunovsky et al. 1996).

Interestingly, the mRNA export competent mutant (W73A) rescued cells toa similar extent as cells overexpressing wildtype 4E. This suggests that4E's rescue function is, at least in part, mediated via its mRNA exportfunction. In contrast, the inactive W56A 4E mutant did not rescue cells,with a similar number of viable cells as the vector controls.

A comparison of vector controls for Akt1−/− cells versus wildtype cellsshowed that serum deprivation of Akt1−/− cells had slightly reducedviability relative to wildtype cells (˜20% versus ˜40%). This reductionin viability was observed in other studies involving serum deprivationof these cells (Chen et al. 2001). Strikingly, neither wildtype 4E northe W73A mutant rescued Akt1−/− cells from apoptosis. In both cases, thenumber of viable cells was around 20%, the same as seen in the vectorcontrols. As a control to demonstrate it is possible to rescue Akt1−/−cells, we examined whether the antiapoptotic f actor, Bcl2 (data notshown), could rescue these cells. Substantially more viable Akt1−/−cells (˜80%) were present upon overexpression of Bcl2, indicating thatthese cells can be rescued. Thus, 4E's survival function, in the contextof serum deprivation, requires the presence of Akt1.

Loss of Akt1 Does Not Impair 4E's mRNA Export or Translation Functions

The results in Akt1−/− cells suggest that one or more biochemicalactivities of 4E could be impaired by the loss of Akt, or that 4Emodulates the expression of target genes involved in activation of theAkt pathway. First, we examined whether 4E dependent mRNA export wasimpaired in Akt1−/− cells compared to wildtype controls (FIG. 29A). Weexamined the nuclear mRNA export of cyclin D1 mRNA by monitoring themRNA content in cytoplasmic versus nuclear fractions using quantitativereal time PCR (qPCR) as we have described previously (Culjkovic et al.2005, Culjkovic et al., 2006). tRNAlys and U6snRNA are shown asfractionation controls for monitoring the quality of cytoplasmic andnuclear fractions respectively as we have reported previously (Culjkovicet al. 2005, Culjkovic et al., 2006) (FIG. 29A). The ratio ofcytoplasmic to nuclear mRNA levels are shown in FIG. 29A. Cyclin D1 mRNAwas chosen as it is the best-described 4E dependent mRNA export target(Rousseau et al. 1996; Culjkovic et al. 2005, Culjkovic et al., 2006).Our results show that overexpression of 4E or the W73A export-competentmutant, promoted cyclin D1 mRNA export in either wildtype or Akt1−/−cells as compared to vector controls. Another 4E dependent mRNA exporttarget, NBS1 (Culjkovic et al. 2005, Culjkovic et al., 2006), gavesimilar results. Second, we examined the possibility that the loss ofAkt1 impaired 4E sensitive translation. We examined the levels of VEGFprotein, a well-established translational target of 4E (Clemens andBommer 1999). Clearly, loss of Akt1 did not impair the ability of 4E topromote VEGF translation relative to vector controls (FIG. 29B, bottom).VEGF mRNA export is not altered by 4E overexpression in either wildtypeor Akt1−/− cells (FIG. 29A). Further, mRNA export of GAPDH and actin areunchanged (FIG. 29B, data not shown). This is consistent with previousstudies showing that VEGF, GAPDH and actin are not export targets of 4E,and that VEGF is a translation target (Clemens and Bommer 1999;Culjkovic et al. 2006).

We examined whether alterations in 4E mRNA export activity led toincreased protein production of cyclin D1, NBS1 and VEGF using westernanalysis in Akt1−/− cells as compared to wildtype controls (FIG. 29B,bottom). Further, overexpression of the W73A mutant (which is competentin export but does not enhance translation) leads to increased cyclin D1and NBS1 protein levels, consistent with their enhanced nuclear mRNAexport, but does not enhance production of VEGF protein levels. Therewas no change in the total levels of cyclin D1, NBS1 or VEGF mRNA asmonitored by qPCR as a function of 4E or mutant overexpression (FIG.29B, top). In summary, the loss of Akt1 does not impair 4E dependentmRNA export or translation of the 4E sensitive transcripts examined.This led us to hypothesize that one (or more) of the mRNA targets of 4Ecould potentiate Akt activation.

The 4E Dependent mRNA Export Target, NBS1, is Required for 4E DependentAkt Activation

Our previous studies demonstrated that the ability of 4E to coordinatelymodulate mRNA export of a wide variety of transcripts contributes to itsproliferative potential (Culjkovic et al. 2005, Culjkovic et al., 2006;Culjkovic et al. 2007). Examination of these mRNA targets revealed apotential mechanism for 4E mediated activation of Akt. 4E overexpressionled to enhanced mRNA export of Nijmegen breakage protein 1 (NBS1)(Culjkovic et al. 2006). Traditionally, NBS1 has been associated withDNA double strand break repair (Karran 2000; Petrini 2000; Costanzo etal. 2001). However, recent studies revealed that elevation of NBS1results in activation of PI3K, and subsequently activation of Akt andits downstream effectors, including S6 (Chen et al. 2005). Consistently,NBS1 overexpression is associated with oncogenic transformation andproliferation in cell culture, and tumorigenesis in xenograft mousemodels (Chen et al. 2005; Yang et al. 2006; Yang et al. 2007). Thus, weexamined the possibility that the ability of 4E to activate Akt relied,at least in part, on its ability to modulate expression of NBS1. 4Eoverexpression led to the upregulation of NBS1 mRNA export, similar tothat observed for cyclin D1 mRNA (FIG. 29B) (Rousseau et al. 1996).Consistent with the ability of 4E to promote the mRNA export of NBS1, 4Eoverexpression correlated with increased levels of NBS1 protein and thiswas independent of the presence or absence of Akt1 (FIG. 29B).

These studies led us to hypothesize that NBS1 is an important effectorof 4E dependent activation of Akt. To determine whether 4E required NBS1for Akt activation, NBS1 was knocked down using siRNA methods. Knockdownwas confirmed by western blot analysis (FIG. 30A). Importantly, siRNAtreatment for NBS1 did not alter expression of 4E or Akt (FIG. 30A), nor4E mRNA targets cyclin D1 or VEGF (data not shown). We observe that uponsiRNA treatment for NBS1, 4E overexpression no longer increasesphosphorylation of Akt at either T308 or S473 as observed by westernanalysis as compared to scrambled siRNA controls (FIG. 30A).

It is possible that knockdown of NBS1 modulates 4E's ability to enhancemRNA export, and thus we postulate could regulate the ability of 4E toactivate Akt in some manner independent of the NBS1-PI3K-Akt axis.Further, we examined mRNA export in 4E overexpressing cells treated withsiRNA for NBS1 (siNBS1) or scrambled controls (scram). Our resultsclearly demonstrate that export of cyclin D1 mRNA is not reduced byknockdown of siNBS1 (data not shown). Thus, cyclin D1 mRNA export isenhanced in 4E overexpressing cells versus vector controls whether ornot these cells were treated with siNBS1 or scrambled controls (data notshown). Consistently, cyclin D1 protein levels are upregulated in 4Eoverexpressing cells relative to controls regardless of siRNAtreatments. Interestingly, export of the remaining NBS1 mRNA was alsoelevated in the presence of 4E wildtype or W73A 4E overexpression, againindicating that the mRNA export pathway is intact in cells treated forsiNBS1 (data not shown). Thus knockdown of NBS1 does not impair either4E dependent mRNA export. Taken together with the previously reporteddata on the effects of NBS1 on PI3K activation (Chen et al. 2005) andthe ability of the PI3K inhibitor LY294002 to inhibit this 4E activity(FIG. 27B), it appears that the requirement for NBS1 in 4E mediatedactivation of Akt is linked to the ability of NBS1 to activate PI3K(probably indirectly) and thus, for PI3K to activate Akt.

4E Requires NBS1 for Apoptotic Rescue of Serum Deprived Fibroblasts

We extended our studies to examine whether the ability of 4E toupregulate NBS1 was required, at least in part, for its apoptotic rescuefunction. Akt wildtype cells were treated with siRNA for NBS1 (siNBS1)or scrambled controls (scram), serum deprived and monitored forapoptosis as a function of 4E overexpression (FIGS. 30B and C).Treatment of stable cell lines with scrambled controls, which requiresthe introduction of lipofectamine, slightly reduced viability of cellsrelative to untreated controls for both serum deprivation (FIG. 28, ˜40%to ˜35% observed here, FIG. 30B) and normal conditions (˜80% relative to˜90% in FIG. 28). Knockdown of NBS1 led to a further reduction inviability of cells that were not serum deprived as well as serumdeprived. This is consistent with previous studies indicating that NBS1is required for viability in mouse models (Zhu et al. 2001; Dumon-Joneset al. 2003).

The most striking result from these studies is that knockdown of NBS1severely impaired the survival activity of 4E (FIGS. 30B and C).Specifically, 4E overexpressing cells treated with scrambled controlswere approximately ˜70% viable relative to vector controls which were˜35% viable. This is a very striking extent of rescue and isapproximately the same extent of rescue (2 fold) as observed in FIG. 28.However, the 4E overexpressing cells treated with siRNA for NBS1, haveonly ˜20% of viable cells, 3 fold less than the scrambled controltreated 4E overexpressing cells which were ˜70% viable. Taken togetherwith the observations that 4E requires Akt for its rescue function andrequires NBS1 to activate Akt, our data strongly suggest that thesurvival function, in this context, of 4E requires its ability toactivate Akt through NBS1.

PML is a Negative Regulator of this 4E Activity

Clearly, the cell has developed mechanisms to control the proliferativeand survival functions of 4E. Our previous studies indicated that thepromyelocytic leukemia protein PML is a potent inhibitor of 4E dependentmRNA export (Cohen et al. 2001; Topisirovic et al. 2003a; Culjkovic etal. 2005, Culjkovic et al., 2006; Culjkovic et al. 2007). The RINGdomain of PML directly interacts with the dorsal surface of 4E(including W73), and through a conformational change, reduces theaffinity of 4E for the m7G cap by over 100 fold (Kentsis et al. 2001).Previous studies indicated that mutations of PML in the RING domain(RING) or of the dorsal surface of 4E (W73A) impaired the PML-4Einteraction and thereby relieved the PML mediated inhibition of 4Edependent mRNA export (Cohen et al. 2001; Kentsis et al. 2001; Culjkovicet al. 2005, Culjkovic et al., 2006; Culjkovic et al. 2007). Thus weexamined the possibility that the PML protein impairs export of NBS1mRNA and thereby, impairs 4E dependent activation of Akt. Further, weutilized out mutants to determine if these effects were dependent on thePML-4E interaction.

PML overexpression suppressed mRNA export of NBS1 relative to vectorcontrols or 4E overexpressing cells (FIG. 31). Consistently, PMLimpaired export of cyclin D1 mRNA relative to vector controls (by actingon endogenous 4E) and relative to cells overexpressing 4E (FIG. 31B). Incells expressing both PML and 4E, PML clearly reduces the export of bothNBS1 and cyclin D1 mRNAs relative to cells overexpressing 4E alone.Consistently, NBS1 and cyclin D1 protein levels are reduced relative tocells overexpressing 4E alone (FIG. 31A). Next, we examined whether PMLreduces 4E dependent Akt activation. Co-expression of PML and 4E led toreduction in phosphorylation of Akt at both T308 and S473 relative tocells overexpressing 4E alone (FIG. 31A). Consistently, PMLoverexpression leads to reduced phosphorylation of S6 as well as BP-1relative to vector or 4E overexpressing cells. Thus, PML impairs 4Edependent Akt activation and subsequent downstream events.

In order to demonstrate that these effects of PML are indeed dependenton its interactions with 4E, we monitored the effects of the PML mutantdeficient in 4E binding (RING). In parallel, we monitored the ability ofPML to suppress the W73A 4E mutant, which cannot bind PML. PMLoverexpression impairs 4E mediated Akt activation whereas PML RINGcannot. Further, PML cannot impair Akt activation mediated by the W73A4E mutant. As expected, the PML RING mutant could not inhibit 4Edependent mRNA export of either NBS1 or cyclin D1 mRNA (PML RING+4Eversus PML+4E; FIG. 31B). Furthermore, wildtype PML could not inhibitthe W73A 4E mutant (PML+W73A 4E versus W73A). Consistently, NBS1 proteinlevels were elevated to a similar extent in the PML+W73A or W73A 4Eexpressing cells (FIG. 31A). Thus, PML requires its ability to directlybind to 4E in order to impair 4E dependent NBS1 mRNA export andsubsequent NBS1 protein levels and Akt activation. Co-expression of PMLRING and 4E or of PML and W73A 4E did not lead to impairment in 4Edependent Akt activation, as observed by western blot for both T308 andS473 Akt sites, relative to cells expressing 4E alone (FIG. 31A).Importantly, expression of PML or PMLRING did not alter the expressionof 4E or Akt. Further, expression of 4E or W73A 4E did not modulate PMLlevels.

We hypothesized that PML should impair 4E dependent rescue of serumdeprived fibroblasts. We monitored apoptosis as described above. Priorto serum starvation, PML and 4E do not appear to impact on viability(FIG. 32). However, in serum deprived cells, PML overexpression resultsin reduced viability relative to vector controls (2 fold, FIG. 32A)consistent with our earlier studies (Borden et al. 1997)). 4Eoverexpressing cells result in enhanced viability versus vector controls(2 fold) and PML (4 fold) expressing cells. In contrast in cellsco-expressing PML and 4E, viability was substantially reduced relativeto 4E overexpressing cells (˜40% versus ˜80%). TUNEL assays yieldedconsistent results (FIG. 32B) Thus, PML impairs 4E mediated apoptoticrescue under serum deprivation conditions.

4E is Positioned to have a Two Tier Effect on Akt Expression

Given that 4E modulates gene expression combinatorially (Culjkovic etal. 2007), we investigated whether other known targets of 4E dependentmRNA export and 4E sensitive translation also acted in Akt signaling.Inspection of previously reported 4E mRNA export targets demonstratedthat this is indeed the case i.e. 4E coordinately upregulated effectorsof the Akt pathway including cyclins A2 (Heron-Milhavet et al. 2006), B1(Lee et al. 2005), and E (Hlobilkova et al. 2006; Kim et al. 2006),c-myc (Ahmed et al. 1997; Chen and Sytkowski 2001), and Mdm2 (Mayo andDonner 2001; Gottlieb et al. 2002), as well as cyclin D1(Muise-Helmericks et al. 1998; Gille and Downward 1999; Takuwa et al.1999) and NBS1 (Chen et al. 2005) (FIG. 5, (Culjkovic et al. 2006)).This list is not inclusive, and as more 4E mRNA targets are identified,it is likely many of these will also be downstream effectors of the Aktpathway. Thus, 4E is positioned to effect Akt pathway at two levels: Aktactivation and upregulation of downstream effectors of Akt.

Discussion

We provide evidence that 4E, via the RNA regulon model, modulates thePI3K/Akt signaling axis, and coordinates its regulation (FIG. 33). Thisis consistent with previous studies which indicated that 4E is a node ina regulon that governs cell cycle progression via coordinatelymodulating expression of genes involved in this process (Culjkovic etal. 2005, Culjkovic et al., 2006). The studies reported here indicatethat 4E, using the same strategies, can enhance survival signaling,enabling 4E to drive proliferation whilst inhibiting apoptosis inimmortalized cell lines. Our studies also suggest that these twobiological effects of 4E overexpression, proliferation and apoptoticrescue, are intrinsically linked through modulation of this RNA regulon.

In this model, 4E coordinately exports mRNAs of protein affected by theAkt pathway, allowing their enhanced production (FIG. 33). CoordinatedmRNA export is achieved by a common element in the 3′UTR of these mRNAs,the 4E-SE. To date, our results indicate that 4E impacts on the Aktpathway at least at two levels. First, 4E acts at the level ofphosphorylation of Akt via enhancing production of the NBS1 protein.NBS1 was shown to be an upstream activator of PI3K by other groups (Chenet al. 2005). NBS1 also activates Atm kinases where it is believed toplay an active role and directly associates with Atm (Karran 2000;Petrini 2000; Viniegra et al. 2005). NBS1 contains a PI3K like bindingdomain, and may use this domain to directly interact with PI3K(Cerosaletti et al. 2006). However, the precise mechanism by which NBS1activates P13K, and thereby Akt, is not yet known. Our report isconsistent with previous studies showing that NBS1 activation is PI3Kdependent since LY294002 impairs this activation (Chen et al. 2005).Interestingly, 4E enhances production of ODC, at both the mRNA exportand translation levels (Rousseau et al. 1996). ODC overexpression canlead to Akt activation independent of PI3K (Hayes et al. 2006),indicating that in some contexts, 4E may be able to activate Aktindependently of the NBS1-PI3K-Akt axis we describe here. Second, 4Eoverexpression leads to increased protein levels for several downstreameffectors of Akt (FIGS. 27 & 31, (Culjkovic et al. 2006)). Thus 4E ispositioned to amplify the effects of Akt survival signaling.

4E overexpression in transgenic mouse models of lymphoma correlates withaggressive disease and the development of tumours which are rapamycinresistant (Wendel et al. 2006). In previous models of Akt signaling, itwas difficult to understand how rapamycin resistance would develop giventhat 4E was thought to be only downstream of Akt. Rapamycin inhibitsmTOR and thus mTOR mediated phosphorylation of BP1. In fact, mTORinhibition leads to Akt activation in some cells and patient specimensdue to the fact that mTOR is part of a negative feedback loop on Aktactivity (O'Reilly et al. 2006). Our data provide a possible molecularbasis for 4E mediated rapamycin resistance. 4E is known to enhancelevels of Pim1 (at both the mRNA export and translation level) (Hooveret al. 1997; Clemens and Bommer 1999; Culjkovic et al. 2006) and Pim1can directly phosphorylate BP1 independently of Akt (Hammerman et al.2005). Thus, 4E can bypass mTOR-rapamycin and directly relieveinhibition via Pim1 mediated phosphorylation of BP1. There are likelyseveral other similar means by which 4E can achieve this result.

The effects of 4E overexpression on BP1 phosphorylation are interestingand suggest that that 4E could be involved in a positive feedback loopwhere it activates its translational activity by indirectly using itsmRNA export activity to increase levels of hyperphosphorylated BP1without changing levels of total BP1 protein. However, translation of 4Esensitive mRNAs is not significantly elevated in BP1−/−, BP2−/− orBP1−/−/BP2−/− cells (Blackshear et al. 1997; Tsukiyama-Kohara et al.2001; Banko et al. 2006; Le Bacquer et al. 2007). Enhancement of theformation of translationally active 4E complexes is estimated to bewithin error of the measurements (˜1.5 fold) (Banko et al. 2006) andenhanced polysomal loading of 4E sensitive mRNAs has not been reportedfor any of these knockout cells. These animals are normal in terms oftheir size with BP1−/− and BP1−/−/BP2−/− having only significant defectsin adipogenesis and the insulin response (Le Bacquer et al. 2007).Interestingly, BP1−/− cells respond to rapamycin (in terms of growtharrest) to the same extent as wildtype controls (Blackshear et al.1997). The phenotype for these mice was predicted to be much moremarked, where it was assumed that mice would be subject to a wide rangeof cancers. This is not the case. These studies suggest that there issignificant redundancy in the factors that regulate 4E. Thus, althoughBP1 phosphorylation is clearly a marker of Akt activation, it is notclear the extent to which BP1 phosphorylation alone can be predictive ofthe translational activity of 4E. Thus, in the case of our studies,although 4E does stimulate BP 1 phosphorylation through Akt activation,the extent of this effect alone on 4E activity will require furtherstudies to unravel. However, these studies do show that 4E mediated Aktactivation leads to the expected signaling events with respect to S6 andBP1.

The cell has clearly developed master control switches to control RNAregulon, in this case in the form of PML, to attenuate the effects of 4E(Culjkovic et al. 2006; Culjkovic et al. 2007). Thus the cell can usePML to shut down this complicated survival network by directly targetingjust one part of the network, 4E. PML is a potent inhibitor of 4E whereit not only inhibits 4E dependent mRNA export, but when in the cytoplasmcan inhibit cap dependent translation as well (Kentsis et al. 2001).Further, the ability of PML to promote apoptosis via inhibiting 4Edependent rescue (FIG. 32) provides the first molecular explanation forprevious observations that the ability of PML to promote apoptosis isindependent of on-going transcription (Quignon et al. 1998), since atthe time of this study the link between PML and 4E was not known. Giventhe model we propose, it is now clear how PML can stimulate apoptosis ina transcriptionally independent manner. Finally, our results areconsistent with recent observations that Akt is more activated in PML−/−cells than in littermate controls (Trotman et al. 2006). Althoughanother mechanism for PML inactivation of Akt was proposed by Trotmanand colleagues, our results do not exclude the possibility that PML actsas a negative regulator of Akt directly and/or indirectly throughinhibition of the 4E regulon. Also, overexpression of PRH, anothernegative regulator of 4E (Topisirovic et al. 2003a), decreasesphosphorylation of Akt (data not shown).

Clearly, other cellular modulators of 4E function, such as En2, HoxA9and 4E-BPs are also positioned to potently modulate this regulon(Sonenberg and Gingras 1998; Topisirovic et al. 2003a; Brunet et al.2005; Topisirovic and Borden 2005). Regulators such as HoxA9 areparticularly potent as HoxA9 stimulates both 4E dependent mRNA exportand 4E dependent translation (Topisirovic et al. 2005).

Further, there are likely feedback loops on this regulon. For instance,c-Myc is an mRNA export and translational target of 4E (Clemens andBommer 1999; Culjkovic et al. 2006). Interestingly, both 4E and NBS1 aredirect transcriptional targets of c-myc (Chiang et al. 2003; Schmidt2004). This provides a model for an interesting positive feedback loopbetween these proteins and Akt activation.

In all, these findings open up to the concept of “oncogene addiction”(Jonkers and Berns 2004; Weinstein and Joe 2006), whereby transformationis dependent upon one or a few genes for the maintenance of a malignantphenotype. 4E could therefore be a suitable candidate for such a role,since we postulate it is a central node in the survival signalingregulon. This points to 4E as a potent therapeutic target. A smallmolecule inhibitor of 4E, ribavirin, a physical mimic of the m7G cap, ispositioned to inhibit this survival signaling network.

Materials and Methods

Constructs. pLINKSV40-PML, pcDNA 4E, MSCV-pgk-GFP-4E WT or mutantexpression constructs were previously described (Cohen et al. 2001;Topisirovic et al. 2003b; Culjkovic et al. 2005; Topisirovic and Borden2005). The PML RING mutant (double point mutation in the RING domain ofPML, required for PML function) was previously described (Borden et al.1998).

Cell culture and Treatments. Cells used were maintained in DMEM with 100units/ml penicillin G sodium and 100 μg/ml streptomycin sulfate (allfrom GibcoBRL), with the addition of: 10% newborn calf serum for MEFAkt1 wt and −/− derived cells; 10% fetal calf serum for Bosc-23 cells;and 10% calf serum with 1 mg/ml G418 (GibcoBRL) and 1 μg/ml puromycin(Sigma) for NIH3T3 derived cells. 4E WT and W56 and W73 mutantretroviral vectors were transiently transfected into Bosc-23 Ecopackaging line (kind gift from Guy Sauvageau), and retroviralsupernatants were used to infect MEF Akt1 wt and Akt1−/− cells (kindgift from Morris Birnbaum). GFP+ cells were isolated using the BDFACSAria cell sorter. 4E and PML stably transfected NIH3T3 cells weregenerated as described (Topisirovic et al. 2002; Topisirovic et al.2003a). For siRNA studies, 4E over-expressing MEF Akt1 wt cells weretransfected with Lipofectamine 2000 (GibcoBRL) and 20 nM siRNA duplexMMS.RNAI.NO13752.2.2 (IDT) according to the manufacturer's instruction.Cells were analyzed 72 h after transfection. LY294002 (LY), used intreatment studies was cell culture grade (Sigma) and used at 50 μM for 1hr.

Western analysis and Antibodies. Western analysis was performed asdescribed (Topisirovic et al. 2002; Topisirovic et al. 2003a), with amodified lysis buffer (40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10mM β-glycerophosphate, 50 mM NaF, 0.5 μM NaVO3, 1% (v/v) Triton X100,supplemented with complete protease inhibitors (all from Sigma)). Inaddition, blots for immuno-phosphoprotein detection were blocked in BSAblocking solution (2% (w/v) BSA (Sigma) in TBST), and primary antibodiesdiluted in BSA blocking solution. Antibodies used for immunoblottingwere from Cell Signaling unless otherwise mentioned: mAb anti-4E (BDPharMingen); mAb anti-PML (5E10 (Stuurman et al. 1992)); pAb anti-NBS1;mAb anti-cyclinD1 (BD PharMingen); pAb anti-VEGF (Santa Cruz); pAbsanti-Akt, anti-Phospho Thr 308 Akt, and mAb anti-Phospho Ser 473 Akt;pAbs anti-S6 and anti-Phospho S6 ribosomal protein; pAbs anti-4E-BP1 andanti-Phospho 4E-BP1; mAb anti-GAPDH (MAB374, Chemicon); mAb anti-β-actin(AC-15, Sigma).

Apoptosis assays. Exponentially growing cell cultures derived from MEFAkt1 wt and −/−, and NIH3T3 cells were shifted to 0.1% serum conditionsfor 18 hrs. For Annexin VAPC (Ann. V, BD Biosciences) and propidiumiodide (PI, Sigma) staining, cells and initial PBS washes werecollected, and treated according to the manufacturer's instructions (BDBiosciences). Stained cells were analysed on a BD LSRII flow cytometer,with early apoptotic cells scored as annexin V positive, PI negative toexclude necrotic cells. Assays were performed in triplicate on at leastthree separate occasions. For TUNEL staining, pre-seeded cells oncoverslips were serum withdrawn, fixed and stained with the In situ CellDeath Detection kit, TMR red (Roche) according to manufacturer'sinstruction, then mounted in Vectorshield with4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc).Fluorescence from several fields was observed using a 20× objective lenson a Zeiss LSM 510 laser scanning confocal microscope.

Cellular fractionation and qPCR. Fractionation and RNA isolation were asdescribed (Lai and Borden 2000; Topisirovic et al. 2002). qPCR analyseswere performed using Sybr Green PCR Master mix (ABI) in Mx3000PTMthermal cycler (Stratagene), and data analyzed with MxPro software(Stratagene). All conditions and primers were described previously(Culjkovic et al. 2005). All calculations were done using the relativestandard curve method described in Applied Biosystems User Bulletin #2and are more precisely described in the corresponding figure legends.

Example 7 Ribavirin Inhibits the Anti-Apoptotic Activity of the 4ERegulon and Rapamycin Activates the 4E Regulon

Growth factor/cytokine signaling pathway via normal physiologicalprocesses and/or oncogenic activation of 4E regulon pathway membersresults in a positive feedback loop which in the case of cancer providesfor increased cancer cell survival and proliferation. 4E overexpressionrescues cell from apoptosis-4E rescue of cells from apoptosis isdependent upon Akt since Akt(−/−) cells cannot be rescued by 4E.Increased 4E is one way of accomplishing such an event but also isAkt-activation, Pim-1 activation and/or over-expression, Cyclin D1overexpression, VEGF/FGF2 overexpression and/or various mixtures ofoverexpression and/or activation of the components of the 4E regulon.Inhibition of 4E activity produces an opposing effect with anticipatedtherapeutic benefit such as through treatment of cells, tumors and/orindividuals with 4E inhibitors as epitomized by Ribavirin and relatedcompounds denoted herein and in the literature including compounds uponwhich the structure of Ribavirin was initially based.

By contrast, application of rapamycin and rapamycin analogsknown/unknown in the art can be expected since they activate Akt viaphosphorylation to (i) increase the rate of growth of human cancers and(ii) to provide a mechanism by which cell survival can be provided andthereby provide a clinical benefit in situations where in directcontrast to the over-activation of the 4E regulon (i.e. cancer) whereactivation of the regulon would be therapeutically beneficial, such asin ischemia reperfusion injury and the like as suggested by Amaravadiand Thompson (J Clin Invest 115 2618, 2005) and demonstrated by Rosenand co-workers (Cancer Research 66: 1500, 2006).

As shown in FIGS. 35 and 36, Akt phosphorylation is required foractivation of Akt. Ribavirin inhibits Akt phosphorylation whileRapamycin increases Akt phosphorylation providing for methods to bothinhibit (Ribavirin and compounds with similar regulon activity modifyingactivity) and stimulate (Rapamycin and additional compounds with similarregulon activity modifying activity) the 4E growth/survival regulon.FIG. 37 shows that Ribavirin blocks 4E mediated apoptotic rescue andRapamycin partially inhibits Ribavirin effect on 4E mediated apoptoticrescue.

Discussion

Inhibition of 4E activity can be anticipated to provide therapeuticbenefit by rescuing cells which have become resistant to apoptosis (forexample cancer cells), thereby providing enhanced therapeutic benefit bytherapeutic regimens (including but not limited to chemotherapy,cytotoxic drugs, radiation and targeted therapeutics) by restoring thecell's sensitivity to apoptosis. Further, co-administration eithersequentially and/or simultaneously of 4E inhibitors such as Ribavirin,Ribavirin analogues or molecules upon which the structure of Ribavirinwas based—together—with radiation chemotherapies and/or targetedchemo/bio-therapeutics will provide for superior clinical benefit.

By contrast administration of Rapamycin alone or together withadditional agents known to promote cell survival will provide forsuperior clinical benefit where promotion of cell survival is desireoussuch as subsequent to ischemia-reperfusion injury and the like.

Further, elevation of components of the 4E regulon can be used toprovide diagnostic insight and determination that the 4E regulon isactivated or inactivated (thereby promoting cell survival and apoptosisrespectively). And in those situations treatment with Ribavirin andcompounds previously designated herein or Ribavirin analogues known inthe art or molecules upon which the structure of Ribavirin was initiallybased/conceived can be used to inhibit the 4E survival regulon therebypromoting apoptosis. By contrast, treatment with Rapamycin, rapamycinanalogues or activators of mTOR can be used to stimulate the 4E survivalregulon thereby promoting cell survival which in opposing clinicalsituations from those described above can also be of clinical benefit

Determination of 4E and 4E targets protein and/or activation levels(i.e. molecules regulated at the mRNA transport and/or translationallevel and/or phosphorylation (directly/indirectly) can each providealone or in combination diagnostic insight into the activation-state ofthe 4E regulon. Determining cases where the administration of Ribavirin(et al) or Rapamycin (et al) will provide for the restoration ofcellular apoptosis or the inhibition of cellular apoptosis,respectively. Conditions where restoration of cellular apoptosisprovides clinical benefit include cancer and proliferative diseases anddisease states in which the 4E regulon is activated abnormally and thelike. Conditions where decreasing cellular apoptosis would provideclinical benefit include ischemia-reprefusion injury and the like

Current cancer chemotherapeutic strategies often do not provide for thecoordinated regulation of biological systems, rather they seek toactivate/inhibit a single target and thereby derive therapeutic benefit.By contrast the coordinated therapeutic modulation of a biologicalsystem is likely to provide superior therapeutic since all components ofa pathway are regulated in concert. Few biological systems provide anavenue for just such a coordinated modulation of a biological system andfewer still have been described in sufficient detail to permit thedevelopment of coordinated therapeutic regulators of such systems.

The current art provides for the coordinate regulation of the 4E regulonvia its therapeutic manipulation. 4E, 4E targets (mRNA transport and/ortranslational regulated molecules) and the kinase activities representimportant participants in the oncogenic process and in human cancers.Ribavirin, a high-affinity inhibitor of 4E, inhibits the 4E regulon in acoordinated fashion. By contrast it appears that Rapamycin coordinatelyup-regulates (at least in part) this pathway.

As noted above both the inhibition of the 4E regulon and its activationprovide therapeutic benefits, inhibition of cell growth andsurvival/resistance to chemotherapeutic agents in the case of cancersand hyperproliferative conditions/disease; and activation of cellgrowth/survival in the case of ischemia reperfusion injury and the like.The ability to coordinately regulate (i.e. inhibit) this pathway via theadministration of micormolar concentrations of Ribavirin can provide awide range of therapeutic benefits in cancer and hyperproliferativeconditions, cancers and tuberous sclerosis to name but two categories.

An alternate to targeting single point/members of biological networks isthe more recent development of multi-kinase inhibitors. These agentshave been developed in an attempt to provide greater therapeuticcoverage of target biological systems. The ability of Ribavirin tocoordinately and selectively regulate the activity of numerous kinaseactivities directly/indirectly far exceeds that capabilities provided byeven the broadest multi-kinase inhibitors that have been developed andto provide superior specificity and selectivity of action. FurtherRibavirin in and of itself is not a kinase inhibitor, rather through itsinhibition of 4E activity it inhibits the transport and/or translationof mRNAs encoding either these important regulatory kinase activities ormodulators/activators thereof. Further still, Ribavirin inhibits thetransport and/or translation of mRNAs encoding molecules that providefor tumor metastasis, angiogenesis and resistance to apoptosis as notedin the FIG. 34.

Clearly Ribavirin's ability to coordinately inhibit the 4E regulonprovides superior regulation and thereby therapeutic benefit inconditions (cancer and hyperproliferative disease) where this regulon'sactivity is in all or partially elevated. It follows directly that inaddition to cancer with elevated 4E that cancers with elevated levels ofone or more of the 4E target molecules, 4E activated molecules and 4Eactivating molecules represent (and many have been so characterizedpreviously) oncogenes. As such it is expected that conditions whereinone or more of the members of the 4E regulon is elevated at the level ofgene expression, protein synthesis or activity) will representconditions where the administration of Ribavirin is anticipated toprovide therapeutic benefit. These are likely to include cancers inwhich while 4E activity is normal 4E targets/activators/effectors areelevated. For example, in prostate cancer Pim-1 kinase is elevated in avast majority of advanced prostate cancers. Owing to the fact that Pim-1is a 4E regulated molecule (both at the mRNA transport and mRNAtranslational levels) is appears likely that administration of Ribavirinto Pim-1 positive prostate cancers will likely provide therapeuticbenefit. Similarly, determination in various cancers which of the 4Eregulon components are elevated or present in an constitutively activeform will serve to define which indications are most likely to respondand derived therapeutic benefit from low-dose Ribavirin administration.Ribavirin provides for coordinated and therapeutic modulation of theregulon including modulation of important survival kinases (Akt andPim-1) but also the therapeutic modulation of Akt/Pim-1 target effectormolecules (most commonly via the reduction of the amount of the targetprotein/effector molecule present in cells) This provides superiorcontrol of this central regulatory mechanism which is involved inregulating cell growth, cell survival and in many many cases theseprocesses in cancerous cells and/or tumors.

Example 8 Studies of Additional Components of the 4E Regulon

HuR Could Modulate the 4E Regulon by Modulating the Levels of 4E mRNAand the Activity of the 4E Protein Directly

In this section, we describe a novel mechanism for upregulation of 4Elevels, whereby 4E mRNA stability is increased through interactions withHuR. HuR is overexpressed in several cancers and can transform cells,thus this link might be particularly relevant to elevation of 4E levelsin some cancers. In our studies, we found that HuR proteinimmunoprecipitates with 4E mRNA (data not shown). HuR protein is knownto modulate cyclin D1 mRNA stability through an interaction with the AREelement in the cyclin D1 mRNA. Thus, cyclin D1 is a positive control forthese assays. Note that the 4E protein does not immunoprecipitate withHuR mRNA (data not shown). Further, 4E overexpression does not changeHuR expression (data not shown). Thus it appears that HuR modulates 4EmRNA stability but that 4E does not modulate gene expression of HuR.

HuR is well established to stabilize transcripts with ARE elements intheir 3′ UTR. Thus we examined whether overexpression of HuR wouldmodulate 4E mRNA levels. As observed by western blot analysis, HuRoverexpression led to increased levels of endogenous cyclin D1 protein.As expected, HuR also increased levels of endogenous cyclin D1 proteinlevels. This is consistent with previous studies showing that HuRstabilizes cyclin D1 mRNA. Consistently, parallel studies demonstratethat siRNA knockdown of HuR leads to reduction in 4E levels (data notshown).

We extended these studies to examine the effects of HuR overexpressionon endogenous 4E mRNA stability using actinomycin D. HuR overexpressionsubstantially stabilizes 4E mRNA but not GAPDH (a negative control)relative to vector controls. Given that HuR binds many mRNAs which arealso downstream targets of 4E mRNA export, we examined whether HuR boundto chimeric IacZ constructs which contain the 4E-SE. LacZ without the4E-SE was used as a control. As expected, HuR did not associate witheither the LacZ4E-SE or LacZ mRNAs (data not shown). This is consistentwith previous observations that HuR associated with ARE elements. Thesestudies suggest that the ARE elements in the HuR sensitive mRNAs aredistinct from the 4E-SE. In this way, HuR and 4E could potentiallyassociate with the same transcripts at the same time (using differentUSER codes), coordinately modulating export and stability.

We show by immunoprecipitation of endogenous proteins that HuR proteinbinds to 4E protein in an RNA dependent manner (data not shown). Theinteraction is observed in both the nuclear and cytoplasmic fractionsindicating that HuR could modulate 4E in both mRNA export andtranslation of sensitive mRNAs. Note that HuR and 4E proteins stillimmunoprecipitate in the presence of heparin, but not in the presence ofRNAse. This indicates that a specific RNA interaction mediates the HuR4E protein protein HuR interaction. Note that 4E does notimmunoprecipitate its own mRNA, thus HuR 4E protein complexes aredistinct from HuR-4E mRNA complexes.

Taken together, our preliminary data for HuR suggests that HuR couldmodulate the 4E regulon by modulating the levels of 4E mRNA and theactivity of the 4E protein directly. Further, HuR stimulates theexpression of a downstream target of 4E, cyclin D1 (and others, seebelow). Thus, HuR is positioned to amplify 4E activity. It is thuspossible, that the previously reported transforming and oncogenicproperties of HuR could be mediated, in part, through its interactionwith 4E mRNA and 4E protein. Thus far, our data suggest that HuR canmodulate 4E levels and activity, but that 4E does not modulateexpression of HuR (data not shown). Indeed, our previous data indicatethat 4E overexpression does not lead to alterations in mRNA stability oftarget mRNAs such as cyclin D1, ODC, or model mRNAs such as IacZ-4E-SE(or IacZ controls). Taken together, these data indicate that a subset ofmRNAs associate with both HuR and 4E proteins. These mRNAs likelycontain at least two distinct non-overlapping USER codes, the ARE (forHuR) and the 4E-SE (for 4E). In this way, 4E and HuR can modulate theexpression of a common set of transcripts and thus, mediate theirbiological effects on cell growth.

Hence, we propose that HuR potentially has a three tier effect on the 4Eregulon: 1. it amplifies the regulon by elevating levels of 4E, 2. itincreases the levels of 4E mRNA export targets through stabilizing thesetranscripts thereby increasing the effectiveness of 4E and 3. itdirectly modulates the function of the 4E via the HuR-4E protein-proteininteraction. Overexpression of HuR itself is known to lead to oncogenictransformation in cell culture, to tumours in xenograft mouse models andis elevated in some human cancers. It seems likely that the oncogenicpotential of HuR may arise, at least in part, through its ability tomodulate the 4E regulon.

Outcome. The mechanism by which 4E expression itself is controlled is asubject that has received very little attention. Our studies indicatethat HuR enhances 4E expression by stabilizing 4E mRNA. This is thefirst time such a mechanism has been proposed for 4E, with previousstudies focusing on enhanced transcription or gene amplification beingthe basis for elevated 4E levels in cancer cells.

Example 9 Human Head and Neck SCC Cell Line Experiment

FaDu cells were grown in culture as described previously and treatedwith Ribavirin for 48 hours prior to preparation of protein extracts andwestern blot analysis. Actin and eIF4E protein levels remain unchangedafter Ribavirin treatment (FIG. 38). By contrast the protein level ofNBS1, Cyclin D1 and ODC (proteins whose mRNAs are eIF4E regulated at thenuclear to cytoplasmic transport level) are decreased by Ribavirintreatment (FIG. 38).

REFERENCES

All publications and patents mentioned herein, including thosereferences listed below, are hereby incorporated by reference in theirentirety as if each individual publication or patent was specificallyand individually incorporated by reference. In case of conflict, thepresent application, including any definitions herein, will control.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations. Suchequivalents are intended to be encompassed by the following claims.

The invention claimed is:
 1. A method of inhibiting 4E activity,inhibiting 4E regulon activity, inducing apoptosis, or inhibitingproliferation of a cell, comprising the step of: contacting the cellwith a prodrug form of a compound of Formula I; wherein the compound ofFormula I is represented by:

wherein: R1 is selected from the group consisting of null, a linear orbranched alkyl, alkenyl, hydrogen, and alkynyl group; R2 is selectedfrom the group consisting of a primary amine, a secondary amine, atertiary amine, an aromatic amine, an amino group and an amido group; R3is selected from the group consisting of NH, oxygen and sulfur; and R4is hydroxyl.
 2. The method of claim 1, wherein R1 is —CH₃ or CH₂CH₃. 3.The method of claim 1, wherein R2 is selected from the group consistingof —NH₂, —NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₃)₂, —NHCH₂CH₂OH,—NHCH₂CH₂CH(OH)CH₃, and —NHCH(CH₂OH)CH₃.
 4. The method of claim 1,wherein said cell is a tumor cell.
 5. The method of claim 1, whereinsaid cell is in a subject.
 6. The method of claim 5, wherein thecompound is administered to the subject and the compound is administeredat levels of between about 0.1 and about 1 mg/kg body weight of saidsubject.
 7. The method of claim 5, wherein the compound is administeredto the subject and the compound is administered at levels of betweenabout 0.01 and about 5 mg/kg body weight per day.
 8. The method of claim1, further comprising contacting the cell or administering to saidsubject a cytotoxic agent.
 9. The method of claim 1, further comprisingcontacting the cell or administering to said subject one or more of thefollowing: biologic, kinase inhibitor, and chemotherapeutic agent. 10.The method of claim 1, wherein 4E activity in the cell is inhibited. 11.The method of claim 1, wherein 4E regulon activity in the cell isinhibited.
 12. The method of claim 1, wherein apoptosis of the cell isinduced.
 13. The method of claim 1, wherein proliferation of the cell isinhibited.